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Abstract:

Methods of detecting influenza, including differentiating between type
and subtype are disclosed, for example to detect, type, and/or subtype an
influenza infection. A sample suspected of containing a nucleic acid of
an influenza virus, is screened for the presence or absence of that
nucleic acid. The presence of the influenza virus nucleic acid indicates
the presence of influenza virus. Determining whether the influenza virus
nucleic acid is present in the sample can be accomplished by detecting
hybridization between an influenza specific probe, influenza type
specific probe, and/or subtype specific probe and an influenza nucleic
acid. Probes and primers for the detection, typing and/or subtyping of
influenza virus are also disclosed. Kits and arrays that contain the
disclosed probes and/or primers also are disclosed.

10. The set of primers of claim 9, wherein the forward and reverse
primers are a pair of primers, and wherein the pair of primers: is
specific for the amplification of influenza A and consist of the nucleic
acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, is specific
for the amplification of influenza subtype H1 and consist of the nucleic
acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, is specific
for the amplification of influenza subtype H3 and consist of the nucleic
acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 13, is specific
for influenza type B and consist of the nucleic acid sequences set forth
in SEQ ID NO: 26 and SEQ ID NO: 28, is specific for influenza subtype
North American H7 and consist of the nucleic acid sequences set forth in
SEQ ID NO: 30 and SEQ ID NO: 31, is specific for influenza subtype
European H7 and consist of the nucleic acid sequences set forth in SEQ ID
NO: 33 and SEQ ID NO: 34, or is specific for influenza subtype Asian H9
and consist of the nucleic acid sequences set forth in SEQ ID NO: 36 and
SEQ ID NO: 37.

11. A kit for detecting an influenza virus nucleic acid in a sample,
comprising: at least one of the probes of claim 1; and instructions for
hybridizing the probe to the influenza virus nucleic acid in a sample.

12. The kit of claim 11, further comprising a primer.

13. A kit for detecting an influenza virus nucleic acid in a sample,
comprising at least one of the probes of claim 1 and a pair of primers;
wherein the kit comprises: a probe consisting of the nucleic acid
sequence shown in SEQ ID NO: 8; and a pair of primers consisting of the
nucleic acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4; a probe
consisting of the nucleic acid sequence shown in SEQ ID NO: 11; and a
pair of primers consisting of the nucleic acid sequence shown in SEQ ID
NO: 9 and SEQ ID NO: 10; a probe consisting of the nucleic acid sequence
shown in SEQ ID NO: 14; and a pair of primers consisting of the nucleic
acid sequence shown in SEQ ID NO: 12 and SEQ ID NO: 13; a probe
consisting of the nucleic acid sequence shown in SEQ ID NO: 19; and a
pair of primers consisting of the nucleic acid sequence shown in SEQ ID
NO: 17 and SEQ ID NO: 18; a probe consisting of the nucleic acid sequence
shown in SEQ ID NO: 29; and a pair of primers consisting of the nucleic
acid sequence shown in SEQ ID NO: 26 and SEQ ID NO: 28; a probe
consisting of the nucleic acid sequence shown in SEQ ID NO: 32; and a
pair of primers consisting of the nucleic acid sequence shown in SEQ ID
NO: 30 and SEQ ID NO: 31; a probe consisting of the nucleic acid sequence
shown in SEQ ID NO: 35; and a pair of primers consisting of the nucleic
acid sequence shown in SEQ ID NO: 33 and SEQ ID NO: 34; a probe
consisting of the nucleic acid sequence shown in SEQ ID NO: 38; and a
pair of primers consisting of the nucleic acid sequence shown in SEQ ID
NO: 36 and SEQ ID NO: 37.

14. A device for detecting an influenza virus nucleic acid in a sample,
comprising a nucleic acid array comprising at least one probe of claim 1.

15. A method for diagnosing an influenza virus infection in a subject
suspected of having an influenza infection comprising: obtaining a sample
comprising nucleic acids from the subject; contacting the sample with one
or more the nucleic acid probes of claim 1; detecting hybridization
between an influenza virus nucleic acid sequence present in the sample
and the probe; and determining that the subject is infected with
influenza virus when hybridization between the influenza virus nucleic
acid sequence present in the sample and the probe is detected.

17. The method of claim 15, further comprising discriminating between an
influenza type A infection and an influenza type B infection.

18. The method of claim 15, further comprising discriminating between an
influenza subtype H1 infection, an influenza subtype H3 infection, an
influenza subtype North American H7 infection, an influenza subtype
European H7 infection, and an influenza subtype Asian H9 infection.

19. A method for detecting an influenza virus nucleic acid in a sample,
comprising: contacting the sample with at least one of the probes of
claim 1; detecting hybridization between the influenza virus nucleic acid
and the probe; and determining that the influenza virus nucleic acid is
present in the sample when hybridization between the influenza virus
nucleic acid and the probe is detected.

20. The method of claim 19, wherein detecting hybridization of the probe
to the influenza virus nucleic acid sequence set forth as: SEQ ID NO: 42,
indicates the presence of influenza A in the sample, SEQ ID NO: 44,
indicates the presence of influenza subtype H1 in the sample, SEQ ID NO:
45, indicates the presence of influenza subtype H3 in the sample, SEQ ID
NO: 43, indicates the presence of influenza type B in the sample, SEQ ID
NO: 48, indicates the presence of influenza subtype North American H7 in
the sample, SEQ ID NO: 49, indicates the presence of influenza subtype
European H7 in the sample, or SEQ ID NO: 50, indicates the presence of
influenza subtype Asian H9 in the sample.

21. The method of claim 19, wherein the probe is labeled.

22. The method of claim 21, wherein the probe is radiolabeled,
fluorescently-labeled, biotin-labeled, enzymatically-labeled, or
chemically-labeled.

23. The method of claim 21, wherein detecting hybridization comprises
detecting a change in signal from the labeled probe during or after
hybridization relative to signal from the label before hybridization.

24. The method of claim 21, wherein the probe is labeled with a
fluorophore.

25. The method of claim 21, wherein the probe is labeled with a
fluorescence quencher.

30. The method according to claim 29, wherein the at least one forward
and reverse primer are a pair of primers, wherein the pair of primers: is
specific for the amplification of influenza A and consist of the nucleic
acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, is specific
for the amplification of influenza subtype H1 and consist of the nucleic
acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, is specific
for the amplification of influenza subtype H3 and consist of the nucleic
acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 13, is specific
for influenza type B and consist of the nucleic acid sequences set forth
in SEQ ID NO: 26 and SEQ ID NO: 28, is specific for influenza subtype
North American H7 and consist of the nucleic acid sequences set forth in
SEQ ID NO: 30 and SEQ ID NO: 31, is specific for influenza subtype
European H7 and consist of the nucleic acid sequences set forth in SEQ ID
NO: 33 and SEQ ID NO: 34, or is specific for influenza subtype Asian H9
and consist of the nucleic acid sequences set forth in SEQ ID NO: 36 and
SEQ ID NO: 37.

31. The method according to claim 19, wherein the sample is a biological
sample obtained from a subject.

32. The method of claim 31, wherein the presence of an influenza virus
nucleic acid in the biological sample indicates the presence of an
influenza virus infection in the biological sample obtained from the
subject.

34. The method of claim 19, wherein the probe is arrayed in a
predetermined array with an addressable location.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.
No. 12/191,186, filed Aug. 13, 2008, now U.S. Pat. No. ______, which is a
continuing application of copending International Application No.
PCT/US2007/003646, filed Feb. 12, 2007, published under PCT Article 21(2)
in English, and claims the benefit of U.S. Provisional Application No.
60/772,806, filed Feb. 13, 2006, all of which applications are
incorporated by reference herein in their entirety.

FIELD

[0002] This disclosure relates to primers and probes for detecting one or
more types or subtypes of influenza virus, as well as kits including the
probes and primers and methods of using the probes and primers.

BACKGROUND

[0003] Influenza virus types A and B are members of the orthomyxoviridae
family of viruses that cause influenza infection. The infective potential
of influenza is frequently underestimated and can result in high
morbidity and mortality rates, especially in elderly persons and in
high-risk patients, such as the very young and immuno-compromised.
Influenza A and B viruses primarily infect the nasopharyngeal and
oropharyngeal cavities and produce highly contagious, acute respiratory
disease that results in significant morbidity and economic costs. Typical
influenza viral infections in humans have a relatively short incubation
period of 1 to 2 days, with symptoms that last about a week including an
abrupt onset of fever, sore throat, cough, headache, myalgia, and
malaise. When a subject is infected with a highly virulent strain of
influenza these symptoms can progress rapidly to pneumonia and in some
circumstances death. Pandemic outbreaks of highly virulent influenza
present a serious risk to human and animal health worldwide.

[0004] The immunodominant antigens present on the surface of influenza
viruses are hemagglutinin (HA or H) and neuraminidase (N). Genetic
reassortment between human and avian influenza viruses can result in a
virus with a novel hemagglutinin of avian origin, against which humans
lack immunity. In the 20th century, the pandemics of 1918, 1957, and
1968 were the result of such antigenic shifts. The avian influenza
outbreaks of the early 21st century caused by H5N1, H7N7, and H9N2
subtype influenza viruses, and their infection of humans have created a
new awareness of the pandemic potential of influenza viruses that
circulate in domestic poultry. The economic impact of a major influenza
pandemic has been estimated to be up to $165 billion in the United States
alone, with as many as 200,000 deaths, 730,000 hospitalizations, 42
million outpatient visits, and 50 million additional illnesses.

[0005] To combat influenza infection, neuraminidase inhibitors have
recently been developed. Clinical studies carried out for the Food and
Drug Administration's (FDA) approval of neuraminidase inhibitors in the
United States showed that successful treatment primarily depends on
prompt treatment after the first clinical symptoms occur. Unfortunately,
it is generally not possible for even experienced medical professionals
to reliably diagnose influenza solely on the basis of the patient's
clinical symptoms because other viruses which infect the nasal or
pharyngeal cavity, such as adenoviruses, parainfluenza viruses, or
respiratory syncitial viruses (RS viruses), cause similar symptoms. To
effectively treat influenza infection it is necessary to begin treatment
as early as possible in the development of the infection, ideally upon
the onset of non-virally specific clinical symptoms.

[0006] A variety of methods have been used to detect influenza viruses
clinically. In one example, influenza viruses are detected by culturing
samples obtained from a subject on mammalian cells such as Madine-Darby
Canine Kidney cells (MDCK). Culturing mammalian cells is costly and time
consuming (taking up to 14 days) and is thus not of immediate relevance
for the diagnosis of the individual patient. Other methods of detection
that have been developed include immunofluorescence assays (IFA), enzyme
immunoassays (EIA), and enzyme-linked immunosorbent assays (ELISA) that
use antibodies specific to influenza virus antigens. Culture and
serological tests require long completion times (5 days to 2 weeks) with
potentially greater exposure of technical personnel to infectious agents.
Immunoassays are generally faster (30 minutes to 4 hours) but often
require substantial sample handling and rely on subjective determination
of results by technical personnel. Furthermore, these tests typically are
not capable of rapidly differentiating between the influenza types and
subtypes, some of which have pandemic potential.

[0007] Hence the need remains for a test that provides sensitive, specific
detection of influenza virus types and subtypes in a relatively short
time, so that diagnosis is completed in sufficient time to permit
effective treatment of an infected person.

SUMMARY

[0008] The present disclosure relates to methods of detecting the presence
of an influenza virus in a sample, such as a biological sample obtained
from a subject. The disclosed methods can be used for diagnosing an
influenza infection in a subject suspected of having an influenza
infection by analyzing a biological specimen from a subject to detect a
broad variety of influenza types and subtypes. Alternatively, the method
can be used to quickly identify particular types and subtypes of
influenza virus, particularly viruses that may be involved in pandemics.
In addition, panels of probes are provided that permit the rapid
evaluation of a subject with an apparent viral illness by quickly
determining whether the illness is caused by a virulent pandemic virus
(such as an H5 virus, for example H5N1). This rapid evaluation involves
ruling out the presence of the pandemic virus (for example by positively
identifying a non-pandemic pathogen such as influenza type B), ruling in
the presence of the pandemic virus (for example by identifying a pandemic
viral pathogen such as an H5 virus, for example H5N1), or a combination
of both.

[0010] The present disclosure also relates to methods of detecting and/or
discriminating between influenza viral types and/or subtypes. These
methods include contacting a sample with a probe that is specific for an
influenza type and/or subtype and detecting the hybridization between the
influenza type and/or subtype specific probe. Detection of hybridization
between an influenza type and/or subtype specific probe and an influenza
nucleic acid indicates that the influenza type and/or subtype is present
in the sample. In some embodiments, the methods include detecting an
influenza viral type and/or subtype. In one example, detecting
hybridization to a nucleic acid sequence at least 95% identical to SEQ ID
NO:8 indicates the presence of influenza type A. In another example,
detecting hybridization to a nucleic acid sequence at least 95% identical
to SEQ ID NO:11 indicates the presence of influenza subtype H1. In
another example, detecting hybridization to a nucleic acid sequence at
least 95% identical to SEQ ID NO:14 indicates the presence of influenza
subtype H3. In another example, detecting hybridization to a nucleic acid
sequence at least 95% identical to SEQ ID NO:19 indicates the presence of
influenza subtype H5. In another example, detecting hybridization to a
nucleic acid sequence at least 95% identical to SEQ ID NO:24 indicates
the presence of influenza subtype H5. In another example, detecting
hybridization to a nucleic acid sequence at least 95% identical to SEQ ID
NO:29 indicates the presence of influenza type B. In another example,
detecting hybridization to a nucleic acid sequence at least 95% identical
to SEQ ID NO:32 indicates the presence of influenza subtype North
American H7. In another example, detecting hybridization to a nucleic
acid sequence at least 95% identical to SEQ ID NO:35 indicates the
presence of influenza subtype European H7. In yet another example,
detecting hybridization to a nucleic acid sequence at least 95% identical
to SEQ ID NO:38 indicates the presence of subtype Asian H9 in the sample.

[0012] In some embodiments, the influenza nucleic acid is amplified using
at least one primer, such as a pair of primers, specific for an influenza
type and/or subtype. In some examples, a primer specific for influenza
type A includes a nucleic acid sequence at least 95% identical to the
nucleic acid sequence set forth as one of SEQ ID NO:3 or SEQ ID NO:4. In
other examples, a primer specific for influenza subtype H1 includes a
nucleic acid sequence at least 95% identical to the nucleic acid sequence
set forth as one of SEQ ID NO:9 or SEQ ID NO:10. In other examples, a
primer specific for influenza subtype H3 includes a nucleic acid sequence
at least 95% identical to the nucleic acid sequence set forth as one of
SEQ ID NO:12 or SEQ ID NO:13. In other examples, a primer specific for
influenza subtype H5 includes a nucleic acid sequence at least 95%
identical to the nucleic acid sequence set forth as one of SEQ ID NO:17
or SEQ ID NO:18. In other examples, a primer specific for influenza
subtype H5 includes a nucleic acid sequence at least 95% identical to the
nucleic acid sequence set forth as one of SEQ ID NO:22 or SEQ ID NO:23.
In other examples, a primer specific for influenza type B includes a
nucleic acid sequence at least 95% identical to the nucleic acid sequence
set forth as one of SEQ ID NO:26 or SEQ ID NO:28. In other examples, a
primer specific for influenza subtype North American H7 includes a
nucleic acid sequence at least 95% identical to the nucleic acid sequence
set forth as one of SEQ ID NO:30 or SEQ ID NO:31. In other examples, a
primer specific for influenza subtype European H7 includes a nucleic acid
sequence at least 95% identical to the nucleic acid sequence set forth as
one of SEQ ID NO:33 or SEQ ID NO:34. In other examples, a primer specific
for influenza subtype Asian H9 includes a nucleic acid sequence at least
95% identical to the nucleic acid sequence set forth as one of SEQ ID
NO:36 or SEQ NO:37.

[0013] Additional methods for detecting, typing, and/or subtyping an
influenza virus in a sample include hybridizing nucleic acids in the
sample to at least one influenza type and/or subtype specific probe
arrayed in a predetermined array with an addressable location.

[0016] The disclosure also provides devices, such as arrays, as well as
kits for detecting, typing, and/or subtyping an influenza virus in a
sample suspected of containing an influenza virus.

[0017] The foregoing and other objects, features, and advantages of the
invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a schematic representation of a generalized procedure for
hybridizing an influenza specific probe to an influenza nucleic acid.

[0019]FIG. 2 is a schematic representation of a generalized procedure for
hybridizing an influenza specific probe to an influenza nucleic acid,
wherein the influenza nucleic acid is initially a double stranded nucleic
acid.

[0020]FIG. 3 is a schematic representation of a generalized procedure for
hybridizing and detecting influenza using an influenza specific
TAQMAN® probe.

[0022]FIG. 5A is a graph of a dilution series of SYBER green binding to
influenza nucleic acids amplified with influenza A specific primers.

[0023]FIG. 5B is a graph of the dissociation curves obtained from the
meting of influenza nucleic acids amplified with influenza A specific
primers as shown in FIG. 5A.

[0024]FIG. 5C is a plot of the Ct values extracted from the graphs shown
in FIG. 5A, as a function of concentration of template nucleic acid
concentration.

[0025] FIG. 6A is a graph of data obtained from rt RT-PCRs run on a
dilution series of influenza nucleic acids using an influenza A specific
probe/primer set.

[0026] FIG. 6B is a plot of the Ct values obtained from the graphs shown
in FIG. 6A, as a function of template nucleic acid concentration.

[0027]FIG. 7 is a graph of the data obtained from a series of rt RT-PCRs
run at annealing temperatures ranging from 50-62.5° C.

[0028]FIG. 8A is a graph of data generated from rt RT-PCRs of a sample
obtained from a subject using the indicated influenza type and subtype
TAQMAN® probes.

[0029]FIG. 8B is a graph of data generated from rt RT-PCRs of a sample
obtained from a subject using the indicated influenza type and subtype
TAQMAN11 probes.

[0030]FIG. 8C is a graph of data generated from rt RT-PCRs of a sample
obtained from a subject using the indicated influenza type and subtype
TAQMAN® probes.

[0031] FIGS. 9A-9F show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza type A M gene (SEQ ID NO: 42) used to design the disclosed
influenza type A specific probes and primers.

[0032] FIGS. 10A-10I show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza type B NS gene (SEQ ID NO: 43) used to design the disclosed
influenza type B specific probes and primers.

[0033] FIGS. 11A-11F show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza subtype H1 HA gene (SEQ ID NO: 44) used to design the
disclosed influenza subtype H1 specific probes and primers.

[0034] FIGS. 12A-12F show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza subtype H3 HA gene (SEQ ID NO: 45) used to design the
disclosed influenza subtype H3 specific probes and primers.

[0035] FIGS. 13A-13I show a table showing the consensus sequence and
variations present in the specified influenza isolates for a region of
the influenza subtype H5 HA gene (SEQ ID NO: 46) used to design the
disclosed influenza subtype H5 specific probes and primers that.

[0036] FIGS. 14A-14L show a table showing the consensus sequence and
variations present in the specified influenza isolates for a region of
the influenza subtype H5 HA gene (SEQ ID NO: 47) used to design the
disclosed influenza subtype H5 specific probes and primers.

[0037] FIGS. 15A-15B show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza subtype North American H7 HA gene (SEQ ID NO: 48) used to
design the disclosed influenza subtype North American H7 specific probes
and primers.

[0038] FIGS. 16A-16C show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza subtype European H7 HA gene (SEQ ID NO: 49) used to design
the disclosed influenza subtype European H7 specific probes and primers.

[0039] FIGS. 17A-17I show a table showing the consensus sequence and
variations present in the specified influenza isolates for the region of
the influenza subtype Asian H9 HA gene (SEQ ID NO: 50) used to design the
disclosed influenza subtype Asian H9 specific probes and primers.

DETAILED DESCRIPTION

I. Explanation of Terms

[0040] Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular biology can
be found in Benjamin Lewin, Genes VII, published by Oxford University
Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular
Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995; and other similar
references.

[0041] As used herein, the singular forms "a," "an," and "the," refer to
both the singular as well as plural, unless the context clearly indicates
otherwise. For example, the term "a probe" includes single or plural
probes and can be considered equivalent to the phrase "at least one
probe."

[0042] As used herein, the term "comprises" means "includes." Thus,
"comprising a probe" means "including a probe" without excluding other
elements.

[0043] It is further to be understood that all base sizes or amino acid
sizes, and all molecular weight or molecular mass values, given for
nucleic acids or polypeptides are approximate, and are provided for
descriptive purposes, unless otherwise indicated. Although many methods
and materials similar or equivalent to those described herein can be
used, particular suitable methods and materials are described below. In
case of conflict, the present specification, including explanations of
terms, will control. In addition, the materials, methods, and examples
are illustrative only and not intended to be limiting.

[0044] To facilitate review of the various embodiments of the invention,
the following explanations of terms are provided:

[0045] Animal: A living multi-cellular vertebrate or invertebrate
organism, a category that includes, for example, mammals and birds. The
term mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects, such as
birds.

[0046] Amplification: To increase the number of copies of a nucleic acid
molecule. The resulting amplification products are called "amplicons."
Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)
refers to use of a technique that increases the number of copies of a
nucleic acid molecule in a sample. An example of amplification is the
polymerase chain reaction (PCR), in which a sample is contacted with a
pair of oligonucleotide primers under conditions that allow for the
hybridization of the primers to a nucleic acid template in the sample.
The primers are extended under suitable conditions, dissociated from the
template, re-annealed, extended, and dissociated to amplify the number of
copies of the nucleic acid. This cycle can be repeated. The product of
amplification can be characterized by such techniques as electrophoresis,
restriction endonuclease cleavage patterns, oligonucleotide hybridization
or ligation, and/or nucleic acid sequencing.

[0048] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and transcriptional regulatory sequences.
cDNA also can contain untranslated regions (UTRs) that are responsible
for translational control in the corresponding RNA molecule. cDNA can be
synthesized in the laboratory by reverse transcription from RNA.

[0049] Change: To become different in some way, for example to be altered,
such as increased or decreased. A detectable change is one that can be
detected, such as a change in the intensity, frequency or presence of an
electromagnetic signal, such as fluorescence. In some examples, the
detectable change is a reduction in fluorescence intensity. In some
examples, the detectable change is an increase in fluorescence intensity.

[0050] Complementary: A double-stranded DNA or RNA strand consists of two
complementary strands of base pairs. Complementary binding occurs when
the base of one nucleic acid molecule forms a hydrogen bond to the base
of another nucleic acid molecule. Normally, the base adenine (A) is
complementary to thymidine (T) and uracil (U), while cytosine (C) is
complementary to guanine (G). For example, the sequence 5'-ATCG-3' of one
ssDNA molecule can bond to 3'-TAGC-5' of another ssDNA to form a dsDNA.
In this example, the sequence 5'-ATCG-3' is the reverse complement of
3'-TAGC-5'.

[0051] Nucleic acid molecules can be complementary to each other even
without complete hydrogen-bonding of all bases of each molecule. For
example, hybridization with a complementary nucleic acid sequence can
occur under conditions of differing stringency in which a complement will
bind at some but not all nucleotide positions.

[0052] Detect: To determine if an agent (such as a signal or particular
nucleotide or amino acid) is present or absent. In some examples, this
can further include quantification. For example, use of the disclosed
probes in particular examples permits detection of a fluorophore, for
example detection of a signal from an acceptor fluorophore, which can be
used to determine if a nucleic acid corresponding to nucleic acid of an
influenza virus is present.

[0053] Electromagnetic radiation: A series of electromagnetic waves that
are propagated by simultaneous periodic variations of electric and
magnetic field intensity, and that includes radio waves, infrared,
visible light, ultraviolet light, X-rays and gamma rays. In particular
examples, electromagnetic radiation is emitted by a laser, which can
possess properties of monochromaticity, directionality, coherence,
polarization, and intensity. Lasers are capable of emitting light at a
particular wavelength (or across a relatively narrow range of
wavelengths), for example such that energy from the laser can excite a
donor but not an acceptor fluorophore.

[0054] Emission or emission signal: The light of a particular wavelength
generated from a fluorophore after the fluorophore absorbs light at its
excitation wavelengths.

[0055] Excitation or excitation signal: The light of a particular
wavelength necessary to excite a fluorophore to a state such that the
fluorophore will emit a different (such as a longer) wavelength of light.

[0056] Fluorophore: A chemical compound, which when excited by exposure to
a particular stimulus such as a defined wavelength of light, emits light
(fluoresces), for example at a different wavelength (such as a longer
wavelength of light).

[0057] Fluorophores are part of the larger class of luminescent compounds.
Luminescent compounds include chemiluminescent molecules, which do not
require a particular wavelength of light to luminesce, but rather use a
chemical source of energy. Therefore, the use of chemiluminescent
molecules (such as aequorin) eliminates the need for an external source
of electromagnetic radiation, such as a laser.

[0059] Other suitable fluorophores include those known to those skilled in
the art, for example those available from Molecular Probes (Eugene,
Oreg.). In particular examples, a fluorophore is used as a donor
fluorophore or as an acceptor fluorophore.

[0060] "Acceptor fluorophores" are fluorophores which absorb energy from a
donor fluorophore, for example in the range of about 400 to 900 nm (such
as in the range of about 500 to 800 nm). Acceptor fluorophores generally
absorb light at a wavelength which is usually at least 10 nm higher (such
as at least 20 nm higher) than the maximum absorbance wavelength of the
donor fluorophore, and have a fluorescence emission maximum at a
wavelength ranging from about 400 to 900 nm. Acceptor fluorophores have
an excitation spectrum which overlaps with the emission of the donor
fluorophore, such that energy emitted by the donor can excite the
acceptor. Ideally, an acceptor fluorophore is capable of being attached
to a nucleic acid molecule.

[0061] In a particular example, an acceptor fluorophore is a dark
quencher, such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular
Probes), BLACK HOLE QUENCHERS (Glen Research), ECLIPSE® Dark Quencher
(Epoch Biosciences), or IOWA BLACK (Integrated DNA Technologies). A
quencher can reduce or quench the emission of a donor fluorophore. In
such an example, instead of detecting an increase in emission signal from
the acceptor fluorophore when in sufficient proximity to the donor
fluorophore (or detecting a decrease in emission signal from the acceptor
fluorophore when a significant distance from the donor fluorophore), an
increase in the emission signal from the donor fluorophore can be
detected when the quencher is a significant distance from the donor
fluorophore (or a decrease in emission signal from the donor fluorophore
when in sufficient proximity to the quencher acceptor fluorophore).

[0062] "Donor Fluorophores" are fluorophores or luminescent molecules
capable of transferring energy to an acceptor fluorophore, thereby
generating a detectable fluorescent signal from the acceptor. Donor
fluorophores are generally compounds that absorb in the range of about
300 to 900 nm, for example about 350 to 800 nm. Donor fluorophores have a
strong molar absorbance coefficient at the desired excitation wavelength,
for example greater than about 103 M-1 cm-1.

[0063] Fluorescence Resonance Energy Transfer (FRET): A spectroscopic
process by which energy is passed between an initially excited donor to
an acceptor molecule separated by 10-100 Å. The donor molecules
typically emit at shorter wavelengths that overlap with the absorption of
the acceptor molecule. The efficiency of energy transfer is proportional
to the inverse sixth power of the distance (R) between the donor and
acceptor (1/R6) fluorophores and occurs without emission of a
photon. In applications using FRET, the donor and acceptor dyes are
different, in which case FRET can be detected either by the appearance of
sensitized fluorescence of the acceptor or by quenching of donor
fluorescence. For example, if the donor's fluorescence is quenched it
indicates the donor and acceptor molecules are within the Forster radius
(the distance where FRET has 50% efficiency, about 20-60 Å), whereas
if the donor fluoresces at its characteristic wavelength, it denotes that
the distance between the donor and acceptor molecules has increased
beyond the Forster radius, such as when a TAQMAN®probe is degraded by
Taq polymerase following hybridization of the probe to a target nucleic
acid sequence or when a hairpin probe is hybridized to a target nucleic
acid sequence. In another example, energy is transferred via FRET between
two different fluorophores such that the acceptor molecule can emit light
at its characteristic wavelength, which is always longer than the
emission wavelength of the donor molecule.

[0064] Examples of oligonucleotides using FRET that can be used to detect
amplicons include linear oligoprobes, such as HybProbes, 5' nuclease
oligoprobes, such as TAQMAN11 probes, hairpin oligoprobes, such as
molecular beacons, scorpion primers and UniPrimers, minor groove binding
probes, and self-fluorescing amplicons, such as sunrise primers.

[0065] Hybridization: The ability of complementary single-stranded DNA or
RNA to form a duplex molecule (also referred to as a hybridization
complex). Nucleic acid hybridization techniques can be used to form
hybridization complexes between a probe or primer and a nucleic acid,
such as an influenza nucleic acid. For example, a probe or primer (such
as any of SEQ ID NOS:3-38) having some homology to an influenza nucleic
acid molecule will form a hybridization complex with an influenza nucleic
acid molecule (such as any of SEQ ID NOS:42-50). Hybridization occurs
between a single stranded probe and a single stranded target nucleic acid
(such as an influenza nucleic acid), as illustrated in FIG. 1. When the
target nucleic acid is initially one strand of a duplex nucleic acid the
duplex must be melted (at least partially) for the probe to hybridize.
This situation is illustrated in FIG. 2.

[0066] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method and the composition and length of the hybridizing nucleic acid
sequences. Generally, the temperature of hybridization and the ionic
strength (such as the Na+ concentration) of the hybridization buffer will
determine the stringency of hybridization. Calculations regarding
hybridization conditions for attaining particular degrees of stringency
are discussed in Sambrook et al., (1989) Molecular Cloning, second
edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and
11). The following is an exemplary set of hybridization conditions and is
not limiting:

[0067] Very High Stringency (detects sequences that share at least 90%
identity)

[0078] The probes and primers disclosed herein can hybridize to influenza
nucleic acids under low stringency, high stringency, and very high
stringency conditions.

[0079] Influenza Virus: Influenza viruses are enveloped negative-sense
viruses belonging to the orthomyxoviridae family. Influenza viruses are
classified on the basis of their core proteins into three distinct types:
A, B, and C. Within these broad classifications, subtypes are further
divided based on the characterization of two antigenic surface proteins
hemagglutinin (HA or H) and neuraminidase (NA or N). While B and C type
influenza viruses are largely restricted to humans, influenza A viruses
are pathogens of a wide variety of species including humans, non-human
mammals, and birds. Periodically, non-human strains, particularly of
avian influenza, have infected human populations, in some cases causing
severe disease with high mortality. Recombination between such avian
strains and human strains in coinfected individuals has given rise to
recombinant influenza viruses to which immunity is lacking in the human
population, resulting in influenza pandemics. Three such pandemics
occurred during the twentieth century (pandemics of 1918, 1957, and 1968)
and resulted in numerous deaths world-wide.

[0080] Influenza viruses have a segmented single-stranded (negative or
antisense) genome. The influenza virion consists of an internal
ribonucleoprotein core containing the single-stranded RNA genome and an
outer lipoprotein envelope lined by a matrix protein. The segmented
genome of influenza consists of eight linear RNA molecules that encode
ten polypeptides. Two of the polypeptides, HA and NA include the primary
antigenic determinants or epitopes required for a protective immune
response against influenza. Based on the antigenic characteristics of the
HA and NA proteins, influenza strains are classified into subtypes. For
example, recent outbreaks of avian influenza in Asia have been
categorized as H5N1, H7N7, and H9N2 based on their HA and NA phenotypes.

[0081] HA is a surface glycoprotein which projects from the lipoprotein
envelope and mediates attachment to and entry into cells. The HA protein
is approximately 566 amino acids in length, and is encoded by an
approximately 1780 base polynucleotide sequence of segment 4 of the
genome. Polynucleotide and amino acid sequences of HA (and other
influenza antigens) isolated from recent, as well as historic, avian
influenza strains can be found, for example in the GENBANK® database
(available on the world wide web at ncbi.nlm.nih.gov/entrez) or the
Influenza Sequence Database of Los Alamos National Laboratories (LANL)
(available on the world wide web at http://www.flu.lanl.gov). For
example, recent avian H1 subtype HA sequences include: AY038014, and
J02144; recent avian H3 subtype HA sequences include: AY531037, M29257,
and U97740; H5 subtype HA sequences include: AY075033, AY075030,
AY818135, AF046097, AF046096, and AF046088; recent H7 subtype HA
sequences include: AJ704813, AJ704812, and Z47199; and, recent avian H9
subtype HA sequences include: AY862606, AY743216, and AY664675.

[0082] In addition to the HA antigen, which is the predominant target of
neutralizing antibodies against influenza, the neuraminidase (NA)
envelope glycoprotein is also a target of the protective immune response
against influenza. NA is an approximately 450 amino acid protein encoded
by an approximately 1410 nucleotide sequence of influenza genome segment
6. Recent pathogenic avian strains of influenza have belonged to the N1,
N7 and N2 subtypes. Exemplary NA polynucleotide and amino acid sequences
include for example, N1: AY651442, AY651447, and AY651483; N7: AY340077,
AY340078 and AY340079; and, N2: AY664713, AF508892, and AF508588.

[0083] The remaining segments of the influenza genome encode the internal
proteins. PB2 is a 759 amino acid polypeptide which is one of the three
proteins which comprise the RNA-dependent RNA polymerase complex. PB2 is
encoded by approximately 2340 nucleotides of the influenza genome segment
1. The remaining two polymerase proteins, PB1, a 757 amino acid
polypeptide, and PA, a 716 amino acid polypeptide, are encoded by a 2341
nucleotide sequence and a 2233 nucleotide sequence (segments 2 and 3),
respectively.

[0084] Segment 5 consists of about 1565 nucleotides encoding an about 498
amino acid nucleoprotein (NP) protein that forms the nucleocapsid.
Segment 7 consists of an about 1027 nucleotide sequence of the M gene,
which encodes the two matrix proteins; an about 252 amino acid M1
protein, and an about 96 amino acid M2 protein, which is translated from
a spliced variant of the M RNA. Segment 8 consists of the NS gene, which
encodes two different non-structural proteins, NS1 and NS2.

[0085] Isolated: An "isolated" biological component (such as a nucleic
acid) has been substantially separated or purified away from other
biological components in which the component naturally occurs, such as
other chromosomal and extrachromosomal DNA, RNA, and proteins. Nucleic
acids that have been "isolated" include nucleic acids purified by
standard purification methods. The term also embraces nucleic acids
prepared by recombinant expression in a host cell as well as chemically
synthesized nucleic acids, such as probes and primers. Isolated does not
require absolute purity, and can include nucleic acid molecules that are
at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or
even 100% isolated.

[0086] Label: An agent capable of detection, for example by
spectrophotometry, flow cytometry, or microscopy. For example, a label
can be attached to a nucleotide, thereby permitting detection of the
nucleotide, such as detection of the nucleic acid molecule of which the
nucleotide is a part. Examples of labels include, but are not limited to,
radioactive isotopes, enzyme substrates, co-factors, ligands,
chemiluminescent agents, fluorophores, haptens, enzymes, and combinations
thereof. Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed for example in Sambrook et
al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John
Wiley & Sons, New York, 1998).

[0087] Nucleic acid (molecule or sequence): A deoxyribonucleotide or
ribonucleotide polymer including without limitation, cDNA, mRNA, genomic
DNA, and synthetic (such as chemically synthesized) DNA or RNA. The
nucleic acid can be double stranded (ds) or single stranded (ss). Where
single stranded, the nucleic acid can be the sense strand or the
antisense strand. Nucleic acids can include natural nucleotides (such as
A, T/U, C, and G), and can also include analogs of natural nucleotides,
such as labeled nucleotides. In one example, a nucleic acid is an
influenza nucleic acid, which can include nucleic acids purified from
influenza viruses as well as the amplification products of such nucleic
acids.

[0088] Nucleotide: The fundamental unit of nucleic acid molecules. A
nucleotide includes a nitrogen-containing base attached to a pentose
monosaccharide with one, two, or three phosphate groups attached by ester
linkages to the saccharide moiety.

[0089] The major nucleotides of DNA are deoxyadenosine 5'-triphosphate
(dATP or A), deoxyguanosine 5'-triphosphate (dGTP or G), deoxycytidine
5'-triphosphate (dCTP or C) and deoxythymidine 5'-triphosphate (dTTP or
T). The major nucleotides of RNA are adenosine 5'-triphosphate (ATP or
A), guanosine 5'-triphosphate (GTP or G), cytidine 5'-triphosphate (CTP
or C) and uridine 5'-triphosphate (UTP or U).

[0090] Nucleotides include those nucleotides containing modified bases,
modified sugar moieties and modified phosphate backbones, for example as
described in U.S. Pat. No. 5,866,336 to Nazarenko et al. (herein
incorporated by reference).

[0092] Examples of modified sugar moieties which may be used to modify
nucleotides at any position on its structure include, but are not limited
to: arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified
component of the phosphate backbone, such as phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a
formacetal or analog thereof.

[0093] Primers: Short nucleic acid molecules, such as a DNA
oligonucleotide, for example sequences of at least 15 nucleotides, which
can be annealed to a complementary target nucleic acid molecule by
nucleic acid hybridization to form a hybrid between the primer and the
target nucleic acid strand. A primer can be extended along the target
nucleic acid molecule by a polymerase enzyme. Therefore, primers can be
used to amplify a target nucleic acid molecule (such as a portion of an
influenza nucleic acid), wherein the sequence of the primer is specific
for the target nucleic acid molecule, for example so that the primer will
hybridize to the target nucleic acid molecule under very high stringency
hybridization conditions.

[0094] The specificity of a primer increases with its length. Thus, for
example, a primer that includes 30 consecutive nucleotides will anneal to
a target sequence with a higher specificity than a corresponding primer
of only 15 nucleotides. Thus, to obtain greater specificity, probes and
primers can be selected that include at least 15, 20, 25, 30, 35, 40, 45,
50 or more consecutive nucleotides.

[0095] In particular examples, a primer is at least 15 nucleotides in
length, such as at least 15 contiguous nucleotides complementary to a
target nucleic acid molecule. Particular lengths of primers that can be
used to practice the methods of the present disclosure (for example, to
amplify a region of an influenza nucleic acid) include primers having at
least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, at least 25, at
least 26, at least 27, at least 28, at least 29, at least 30, at least
31, at least 32, at least 33, at least 34, at least 35, at least 36, at
least 37, at least 38, at least 39, at least 40, at least 45, at least
50, or more contiguous nucleotides complementary to the target nucleic
acid molecule to be amplified, such as a primer of 15-60 nucleotides,
15-50 nucleotides, or 15-30 nucleotides.

[0098] Probe: A probe comprises an isolated nucleic acid capable of
hybridizing to a target nucleic acid (such as an influenza nucleic acid).
A detectable label or reporter molecule can be attached to a probe.
Typical labels include radioactive isotopes, enzyme substrates,
co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and
enzymes.

[0099] Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed, for example, in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989) and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley-Intersciences
(1987).

[0100] In a particular example, a probe includes at least one fluorophore,
such as an acceptor fluorophore or donor fluorophore. For example, a
fluorophore can be attached at the 5'- or 3'-end of the probe. In
specific examples, the fluorophore is attached to the base at the 5'-end
of the probe, the base at its 3'-end, the phosphate group at its 5'-end
or a modified base, such as a T internal to the probe.

[0101] Probes are generally at least 20 nucleotides in length, such as at
least 20, at least 21, at least 22, at least 23, at least 24, at least
25, at least 26, at least 27, at least 28, at least 29, at least 30, at
least 31, at least 32, at least 33, at least 34, at least 35, at least
36, at least 37, at least 38, at least 39, at least 40, at least 41, at
least 42, at least 43, at least 44, at least 45, at least 46, at least
47, at least 48, at least 49, at least 50 at least 51, at least 52, at
least 53, at least 54, at least 55, at least 56, at least 57, at least
58, at least 59, at least 60, or more contiguous nucleotides
complementary to the target nucleic acid molecule, such as 20-60
nucleotides, 20-50 nucleotides, 20-40 nucleotides, or 20-30 nucleotides.

[0102] Polymerizing agent: A compound capable of reacting monomer
molecules (such as nucleotides) together in a chemical reaction to form
linear chains or a three-dimensional network of polymer chains. A
particular example of a polymerizing agent is polymerase, an enzyme which
catalyzes the 5' to 3' elongation of a primer strand complementary to a
nucleic acid template. Examples of polymerases that can be used to
amplify a nucleic acid molecule include, but are not limited to the E.
coli DNA polymerase I, specifically the Klenow fragment which has 3' to
5' exonuclease activity, Taq polymerase, reverse transcriptase (such as
HIV-1 RT), E. coli RNA polymerase, and wheat germ RNA polymerase II.

[0103] The choice of polymerase is dependent on the nucleic acid to be
amplified. If the template is a single-stranded DNA molecule, a
DNA-directed DNA or RNA polymerase can be used; if the template is a
single-stranded RNA molecule, then a reverse transcriptase (such as an
RNA-directed DNA polymerase) can be used.

[0104] Quantitating a nucleic acid molecule: Determining or measuring a
quantity (such as a relative quantity) of nucleic acid molecules present,
such as the number of amplicons or the number of nucleic acid molecules
present in a sample. In particular examples, it is determining the
relative amount or actual number of nucleic acid molecules present in a
sample.

[0105] Quenching of fluorescence: A reduction of fluorescence. For
example, quenching of a fluorophore's fluorescence occurs when a quencher
molecule (such as the fluorescence quenchers listed above) is present in
sufficient proximity to the fluorophore that it reduces the fluorescence
signal (for example, prior to the binding of a probe to an influenza
nucleic acid sequence, when the probe contains a fluorophore and a
quencher).

[0106] Real-time PCR: A method for detecting and measuring products
generated during each cycle of a PCR, which are proportionate to the
amount of template nucleic acid prior to the start of PCR. The
information obtained, such as an amplification curve, can be used to
determine the presence of a target nucleic acid (such as an influenza
nucleic acid) and/or quantitate the initial amounts of a target nucleic
acid sequence. In some examples, real time PCR is real time reverse
transcriptase PCR (rt RT-PCR).

[0107] In some examples, the amount of amplified target nucleic acid (such
as an influenza nucleic acid) is detected using a labeled probe, such as
a probe labeled with a fluorophore, for example a TAQMAN® probe. In
this example, the increase in fluorescence emission is measured in real
time, during the course of the RT-PCR. This increase in fluorescence
emission is directly related to the increase in target nucleic acid
amplification (such as influenza nucleic acid amplification). In some
examples, the change in fluorescence (dRn) is calculated using the
equation dRn=Rn+-Rn.sup.-, with Rn+ being the fluorescence
emission of the product at each time point and Rn.sup.- being the
fluorescence emission of the baseline. The dRn values are plotted against
cycle number, resulting in amplification plots for each sample as
illustrated in FIG. 4. With reference to FIG. 4, the threshold value (Ct)
is the PCR cycle number at which the fluorescence emission (dRn) exceeds
a chosen threshold, which is typically 10 times the standard deviation of
the baseline (this threshold level can, however, be changed if desired).

[0108] Sample: A sample, such as a biological sample, is a sample obtained
from a plant or animal subject. As used herein, biological samples
include all clinical samples useful for detection influenza infection in
subjects, including, but not limited to, cells, tissues, and bodily
fluids, such as: blood; derivatives and fractions of blood, such as
serum; extracted galls; biopsied or surgically removed tissue, including
tissues that are, for example, unfixed, frozen, fixed in formalin and/or
embedded in paraffin; tears; milk; skin scrapes; surface washings; urine;
sputum; cerebrospinal fluid; prostate fluid; pus; bone marrow aspirates;
bronchoalveolar levage; tracheal aspirates; sputum; nasopharyngeal
aspirates; oropharyngeal aspirates; and saliva. In particular
embodiments, the biological sample is obtained from an animal subject,
such as in the form of bronchoalveolar levage, tracheal aspirates,
sputum, nasopharyngeal aspirates, oropharyngeal aspirates, and saliva.

[0109] Sequence identity/similarity: The identity/similarity between two
or more nucleic acid sequences, or two or more amino acid sequences, is
expressed in terms of the identity or similarity between the sequences.
Sequence identity can be measured in terms of percentage identity; the
higher the percentage, the more identical the sequences are. Homologs or
orthologs of nucleic acid or amino acid sequences possess a relatively
high degree of sequence identity/similarity when aligned using standard
methods.

[0111] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et
al., J. Mol. 215:403-10, 1990) is available from several sources,
including the National Center for Biological Information (NCBI, National
Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and
on the Internet, for use in connection with the sequence analysis
programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to
compare nucleic acid sequences, while blastp is used to compare amino
acid sequences. Additional information can be found at the NCBI web site.

[0112] Once aligned, the number of matches is determined by counting the
number of positions where an identical nucleotide or amino acid residue
is present in both sequences. The percent sequence identity is determined
by dividing the number of matches either by the length of the sequence
set forth in the identified sequence, or by an articulated length (such
as 100 consecutive nucleotides or amino acid residues from a sequence set
forth in an identified sequence), followed by multiplying the resulting
value by 100. For example, a nucleic acid sequence that has 1166 matches
when aligned with a test sequence having 1554 nucleotides is 75.0 percent
identical to the test sequence (1166/1554*100=75.0). The percent sequence
identity value is rounded to the nearest tenth. For example, 75.11,
75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16,
75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will
always be an integer. In another example, a target sequence containing a
20-nucleotide region that aligns with 20 consecutive nucleotides from an
identified sequence as follows contains a region that shares 75 percent
sequence identity to that identified sequence (i.e., 15/20*100=75).

##STR00001##

[0113] One indication that two nucleic acid molecules are closely related
is that the two molecules hybridize to each other under stringent
conditions. Stringent conditions are sequence-dependent and are different
under different environmental parameters.

[0114] The nucleic acid probes and primers disclosed herein are not
limited to the exact sequences shown, as those skilled in the art will
appreciate that changes can be made to a sequence, and not substantially
affect the ability of the probe or primer to function as desired. For
example, sequences having at least 80%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% sequence identity
to any of SEQ ID NOS: 3-38 are provided herein. One of skill in the art
will appreciate that these sequence identity ranges are provided for
guidance only; it is possible that probes and primer can be used that
fall outside these ranges.

[0115] Signal: A detectable change or impulse in a physical property that
provides information. In the context of the disclosed methods, examples
include electromagnetic signals such as light, for example light of a
particular quantity or wavelength. In certain examples, the signal is the
disappearance of a physical event, such as quenching of light.

[0116] TAQMAN® probes: As illustrated in FIG. 3, a linear
oligonucleotide probe with a 5' reporter fluorophore such as
6-carboxyfluorescein (FAM) and a 3' quencher fluorophore, such as
BLACKHOLE QUENCHER® 1 (BHQ®1). In the intact TAQMAN® probe,
energy is transferred (via FRET) from the short-wavelength fluorophore to
the long-wavelength fluorophore on the other end, quenching the
short-wavelength fluorescence. After hybridization, the probe is
susceptible to degradation by the endonuclease activity of a processing
Taq polymerase. Upon degradation, FRET is interrupted, increasing the
fluorescence from the short-wavelength fluorophore and decreasing
fluorescence from the long-wavelength fluorophore.

[0117] Target nucleic acid molecule: A nucleic acid molecule whose
detection, quantitation, qualitative detection, or a combination thereof,
is intended. The nucleic acid molecule need not be in a purified form.
Various other nucleic acid molecules can also be present with the target
nucleic acid molecule. For example, the target nucleic acid molecule can
be a specific nucleic acid molecule (which can include RNA such as viral
RNA), the amplification of which is intended. Purification or isolation
of the target nucleic acid molecule, if needed, can be conducted by
methods known to those in the art, such as by using a commercially
available purification kit or the like. In one example, a target nucleic
molecule is an influenza nucleic acid sequence.

II. Overview of Several Embodiments

[0118] Recent increased circulation of highly pathogenic avian influenza,
such as H5N1, in avian populations together with sporadic human
infections of highly pathogenic avian influenza has raised serious
concerns about the pandemic threat of these viruses. The need exists for
methods to rapidly detect and identify influenza viruses, for example to
rapidly diagnose or determine the pandemic potential of viral samples,
such as those obtained from a subject infected or believed to be infected
with an influenza virus.

[0119] Disclosed herein are methods for the universal detection of all
influenza type A and type B viruses as well as for the identification of
the HA genes of influenza A viruses of human health significance
including contemporary human H1 and H3, as well as Asian avian H5,
Eurasian H7, North American H7, and Asian H9 viruses. The methods have
been developed in one embodiment with a unique set of nucleic acid probes
and/or primers that are surprisingly effective at detecting and
discriminating between influenza type A, and type B and subtypes H1, H3,
Asian avian H5, North American avian H7, European avian H7, and Asian
avian H9 using a variety of conditions. This ability to rapidly screen
and identify a virus from among these diverse groups is a significant
public health advantage.

[0120] As disclosed herein, using sequence alignments of all known
influenza viral sequences available, previously unknown regions of high
sequence homology were discovered amongst the individual influenza viral
types and subtypes. These regions were used to create the consensus
sequences shown in FIGS. 9-17. Using these highly homologous regions as a
starting point the disclosed probes and primers were designed such that
they were surprisingly effective at recognizing genetically diverse
influenza isolates within distinct viral types and/or subtypes. Because
of the pandemic potential of influenza subtype Asian avian H5, two
regions of the H5 HA gene regions of high sequence homology used to
design redundant primers and probes.

[0123] In several embodiments, the probe is influenza type specific. An
influenza type specific probe is capable of hybridizing under stringent
conditions (such as high stringency, or very high stringency conditions)
to an influenza virus nucleic acid from a specific influenza type, such
as influenza type A or type B. For example, a probe that is type specific
for influenza type A (such as specific for an influenza type A M gene
sequence, for example the nucleic acid sequence set forth as SEQ ID
NO:42) is not type specific for influenza type B. Likewise, a probe that
is type specific for influenza type B (such as specific for an influenza
type B NS gene sequence, for example the nucleic acid sequence set forth
as SEQ ID NO:43) is not type specific for influenza type A. In other
words a nucleic acid probe that specifically hybridizes to an influenza
type A nucleic acid (such as a nucleic acid that is at least a portion of
the M gene from influenza type A) does not hybridize to an influenza type
B nucleic acid; such nucleic acids would be type specific probes for
influenza type A. Conversely, a nucleic acid probe that specifically
hybridizes to an influenza type B nucleic acid (such as a nucleic acid
that is at least a portion of the NS gene from influenza type B) does not
hybridize to an influenza type A nucleic acid; such nucleic acids would
be type specific probes for influenza type B. Thus, type specific probes
can be used to discriminate the presence of influenza type A from
influenza type B, or the converse. In some embodiments, the probe is
capable of hybridizing under very high stringency conditions to a nucleic
acid from influenza A, for example to an influenza type A nucleic acid
from the M gene of influenza type A set forth as SEQ ID NO:42. In some
embodiments, the probe is capable of hybridizing under very high
stringency conditions to a nucleic acid from influenza B, for example to
an influenza type B nucleic acid from the NS gene of influenza type B set
forth as SEQ ID NO:43.

[0124] In some embodiments, the probe is specific for an influenza type A
sequence. In a specific example, a probe specific for an influenza type A
nucleic acid includes a nucleic acid sequence at least 95% identical to
SEQ ID NO:8. In some embodiments, the probe is specific for an influenza
type B sequence. In a specific example, a probe specific for an influenza
type B nucleic acid includes a nucleic acid sequence at least 95%
identical to SEQ ID NO:29.

[0125] In several embodiments, the probe is influenza subtype specific. An
influenza subtype specific probe is capable of hybridizing under
stringent conditions (such as high stringency, or very high stringency
conditions) to an influenza virus nucleic acid from a specific influenza
subtype, such as influenza subtype H1, H3, H5, North American H7,
European H7, or Asian H9. Subtype specific probes can be used to detect
the presence of and differentiate between the various influenza subtypes.
Such probes are specific for one influenza subtype, for example specific
for an influenza HA sequence that is subtype specific, such as an H1, H3,
H5, North American H7, European H7, or Asian H9 sequence. In some
examples, a probe that is subtype specific for influenza subtype H1 is
not subtype specific for influenza subtype H3, H5, H7 (North American or
European), or Asian H9. In another example, a probe that is subtype
specific for influenza subtype H3 is not subtype specific for influenza
subtype H1, H5, H7 (North American or European), or Asian H9. In another
example, a probe that is subtype specific for influenza subtype H5 is not
subtype specific for influenza subtype H1, H3, H7 (North American or
European), or Asian H9. In another example, a probe that is subtype
specific for influenza subtype North American H7 is not subtype specific
for influenza subtype H1, H3, H5, European H7, or Asian H9. In another
example, a probe that is subtype specific for influenza subtype European
H7 is not subtype specific for influenza subtype H1, H3, H5, North
American H7, or Asian H9. In yet another example, a probe that is subtype
specific for influenza subtype Asian 1-19 is not subtype specific for
influenza subtype H1, H3, H15, or H7 (North American or European). To put
it another way a nucleic acid probe that specifically hybridizes to an
influenza subtype H1 nucleic acid does not hybridize to an influenza
subtype H3 or any other subtype nucleic acid, such nucleic acids would be
type specific probes for influenza type H1. One of skill in the art would
understand that the same trend would hold for the other subtype specific
probes.

[0126] In some embodiments, the probe is specific for an influenza subtype
H1 sequence, such as the nucleic acid sequence set forth as SEQ ID NO:44.
In a specific example, a probe specific for an influenza subtype H1
nucleic acid includes a nucleic acid sequence at least 95% identical to
SEQ ID NO:11. In some embodiments, the probe is specific for an influenza
subtype H3 sequence, such as the nucleic acid sequence set forth as SEQ
ID NO:45. In a specific example, a probe specific for an influenza
subtype H3 nucleic acid includes a nucleic acid sequence at least 95%
identical to SEQ ID NO:14. In some embodiments, the probe is specific for
an influenza subtype H5 sequence, such as the nucleic acid sequence set
forth as SEQ ID NO:46. In a specific example, a probe specific for an
influenza subtype H5 nucleic acid includes a nucleic acid sequence at
least 95% identical to SEQ ID NO:19. In another specific example, a probe
specific for an influenza subtype H5 nucleic acid includes a nucleic acid
sequence at least 95% identical to SEQ ID NO:24. In some embodiments, the
probe is specific for an influenza subtype North American H7 sequence,
such as the nucleic acid sequence set forth as SEQ ID NO:48. In a
specific example, a probe specific for an influenza subtype North
American H7 nucleic acid includes a nucleic acid sequence at least 95%
identical to SEQ ID NO:32. In some embodiments, the probe is specific for
an influenza subtype European H7 sequence, such as the nucleic acid
sequence set forth as SEQ ID NO:49. In a specific example, a probe
specific for an influenza subtype European H7 nucleic acid includes a
nucleic acid sequence at least 95% identical to SEQ ID NO:32. In some
embodiments, the probe is specific for an influenza subtype Asian H9
sequence, such as the nucleic acid sequence set forth as SEQ ID NO:50. In
a specific example, a probe specific for an influenza subtype Asian H9
nucleic acid includes a nucleic acid sequence at least 95% identical to
SEQ ID NO:38.

[0127] In some embodiments, the probe is detectably labeled, either with
an isotopic or non-isotopic label, alternatively the target nucleic acid
(such as an influenza nucleic acid) is labeled. Non-isotopic labels can,
for instance, comprise a fluorescent or luminescent molecule, biotin, an
enzyme or enzyme substrate or a chemical. Such labels are preferentially
chosen such that the hybridization of the probe with target nucleic acid
(such as an influenza nucleic acid) can be detected. In some examples,
the probe is labeled with a fluorophore. Examples of suitable fluorophore
labels are given above. In some examples, the fluorophore is a donor
fluorophore. In other examples, the fluorophore is an accepter
fluorophore, such as a fluorescence quencher. In some examples, the probe
includes both a donor fluorophore and an accepter fluorophore.
Appropriate donor/acceptor fluorophore pairs can be selected using
routine methods. In one example, the donor emission wavelength is one
that can significantly excite the acceptor, thereby generating a
detectable emission from the acceptor. In some examples, the probe is
modified at the 3'-end to prevent extension of the probe by a polymerase.

[0128] In particular examples, the acceptor fluorophore (such as a
fluorescence quencher) is attached to the 3' end of the probe and the
donor fluorophore is attached to a 5' end of the probe. In another
particular example, the acceptor fluorophore (such as a fluorescence
quencher) is attached to a modified nucleotide (such as a T) and the
donor fluorophore is attached to a 5' end of the probe.

[0131] In several embodiments, the primer is influenza type specific. An
influenza type specific primer is capable of hybridizing under stringent
conditions (such as high stringency, or very high stringency conditions)
to an influenza virus nucleic acid from a specific influenza type, such
as influenza type A or type B. For example, a primer that is type
specific for influenza type A is not type specific for influenza type B.
Likewise, a primer that is type specific for influenza type B is not type
specific for influenza type A. In other words a nucleic acid primer that
specifically hybridizes to an influenza type A nucleic acid (such as a
nucleic acid that is at least a portion of the M gene from influenza type
A, for example the nucleic acid sequence set forth as SEQ ID NO:42) does
not hybridize to an influenza type B nucleic acid, such nucleic acids
would be type specific primers for influenza type A. Conversely, a
nucleic acid primer that specifically hybridizes to an influenza type B
nucleic acid (such as a nucleic acid that is at least a portion of the NS
gene from influenza type B, for example the nucleic acid sequence set
forth as SEQ ID NO:43) does not hybridize to an influenza type A nucleic
acid, such nucleic acids would be type specific primers for influenza
type A. Thus, type specific primers can be used to specifically amplify a
nucleic acid from influenza type A or from influenza type B, but not
both. In some embodiments, the primer is capable of hybridizing under
very high stringency conditions to a nucleic acid from influenza A, for
example to an influenza type A nucleic acid from the M gene of influenza
type A set forth as SEQ ID NO:42. In some embodiments, the primer is
capable of hybridizing under very high stringency conditions to a nucleic
acid from influenza B, for example to an influenza type B nucleic acid
from the NS gene of influenza type B set forth as SEQ ID NO:43.

[0132] In some embodiments, the primer is specific for an influenza type A
sequence, such as an influenza type A M gene sequence. In a specific
example, a primer specific for an influenza type A nucleic acid includes
a nucleic acid sequence at least 95% identical to SEQ ID NO:3 or SEQ ID
NO:4. In some embodiments, the primer is specific for an influenza type B
sequence, such as an influenza type B NS gene sequence. In a specific
example, a primer specific for an influenza type B nucleic acid includes
a nucleic acid sequence at least 95% identical to SEQ ID NO:26 or SEQ ID
NO:28.

[0133] In several embodiments, the primer is influenza subtype specific.
An influenza subtype specific primer is capable of hybridizing under
stringent conditions (such as high stringency, or very high stringency
conditions) to an influenza virus nucleic acid from a specific influenza
subtype, such as influenza subtype H1, H3, H5, North American H7,
European H7 or Asian H9. Such primers are specific for one influenza
subtype, for example specific for an influenza HA sequence that is
subtype specific, such as an H1, H3, H5, North American H7, European H7
or Asian H9 HA nucleic acid sequence. Subtype specific primers can be
used to amplify sequences specific to the various influenza subtypes. In
one example, a primer that is subtype specific for influenza subtype H1
is not subtype specific for influenza subtype H3, H5, H7 (North American
or European), or Asian H9. A primer that is subtype specific for
influenza subtype H3 is not subtype specific for influenza subtype H1,
H5, H7 (North American or European), or Asian H9. A primer that is
subtype specific for influenza subtype H5 is not subtype specific for
influenza subtype H1, H3, H7 (North American or European), or Asian H9. A
primer that is subtype specific for influenza subtype North American H7
is not subtype specific for influenza subtype H1, H3, H5, European H7, or
Asian H9. A primer that is subtype specific for influenza subtype
European H7 is not subtype specific for influenza subtype H1, H3, H5,
North American H7, or Asian H9. A primer that is subtype specific for
influenza subtype Asian H9 is not subtype specific for influenza subtype
H1, H3, H5, or H7 (North American or European). To put it another way a
nucleic acid primer that specifically hybridizes to an influenza subtype
H1 nucleic acid does not hybridize to an influenza subtype H3 or any
other subtype nucleic acid, such nucleic acids would be type specific
primers for influenza type H1. One of skill in the art would understand
that this trend holds for the other subtype specific primers.

[0134] In some embodiments, the primer is specific for an influenza
subtype H1 sequence, such as the nucleic acid sequence set forth as SEQ
ID NO:44. In a specific example, a primer specific for an influenza
subtype H1 nucleic acid includes a nucleic acid sequence at least 95%
identical to SEQ ID NO:9 or SEQ ID NO:10. In some examples, the primer is
specific for an influenza subtype H3 sequence, such as the nucleic acid
sequence set forth as SEQ ID NO:45. In a specific example, a primer
specific for an influenza subtype H3 nucleic acid includes a nucleic acid
sequence at least 95% identical to SEQ ID NO:12 or SEQ ID NO:13. In some
examples, the primer is specific for an influenza subtype H5 sequence,
such as the nucleic acid sequence set forth as SEQ ID NO:46. In a
specific example, a primer specific for an influenza subtype H5 nucleic
acid includes a nucleic acid sequence at least 95% identical to SEQ ID
NO:17 or SEQ ID NO:18. In a specific example, a primer specific for an
influenza subtype H5 nucleic acid includes a nucleic acid sequence at
least 95% identical to SEQ ID NO:22 or SEQ ID NO:23. In some examples,
the primer is specific for an influenza subtype North American H7
sequence, such as the nucleic acid sequence set forth as SEQ ID NO:48. In
a specific example, a primer specific for an influenza subtype North
American H7 nucleic acid includes a nucleic acid sequence at least 95%
identical to SEQ ID NO:30 or SEQ ID NO:31. In some examples, the primer
is specific for an influenza subtype European H7 sequence, such as the
nucleic acid sequence set forth as SEQ ID NO:49. In a specific example, a
primer specific for an influenza subtype European H7 nucleic acid
includes a nucleic acid sequence at least 95% identical to SEQ ID NO:33
or SEQ ID NO:34. In some examples, the primer is specific for an
influenza subtype Asian H9 sequence, such as the nucleic acid sequence
set forth as SEQ ID NO:50. In a specific example, a primer specific for
an influenza subtype Asian H9 nucleic acid includes a nucleic acid
sequence at least 95% identical to SEQ ID NO:36 or SEQ ID NO:38.

[0135] In certain embodiments the primers are a set of primers, such as a
pair of primers, capable of hybridizing to and amplifying an influenza
nucleic acid. Such a set primers comprises at least one forward primer
and a least one reverse primer, where the primers are specific for the
amplification of an influenza type or subtype nucleic acid. In some
examples, the set of primers includes a pair of primers that is specific
for the amplification of influenza type A, type B, subtype H1, subtype
H3, subtype H5, subtype North American H7, subtype European H7, or
subtype Asian H9.

[0136] In certain examples, the pair of primers is specific for the
amplification of an influenza type A nucleic acid and includes a forward
primer at least 95% identical to SEQ ID NO:3 and a reverse primer at
least 95% identical to SEQ ID NO:4. In other examples, the pair of
primers is specific for the amplification of an influenza subtype H1 and
includes a forward primer at least 95% identical to SEQ ID NO:9 and a
reverse primer at least 95% identical to SEQ ID NO:10. In other examples,
the pair of primers is specific for the amplification of an influenza
subtype H3 and includes a forward primer at least 95% identical to SEQ ID
NO:12 and a reverse primer at least 95% identical to SEQ ID NO:13. In
other examples, the pair of primers is specific for the amplification of
an influenza subtype H5 and includes a forward primer at least 95%
identical to SEQ ID NO:17 and a reverse primer at least 95% identical to
SEQ ID NO:18. In other examples, the pair of primers is specific for the
amplification of an influenza subtype H5 and includes a forward primer at
least 95% identical to SEQ ID NO:22 and a reverse primer at least 95%
identical to SEQ ID NO:23. In other examples, the pair of primers is
specific for the amplification of an influenza subtype type B and
includes a forward primer at least 95% identical to SEQ ID NO:26 and a
reverse primer at least 95% identical to SEQ ID NO:28. In other examples,
the pair of primers is specific for the amplification of an influenza
subtype North American H7 and includes a forward primer at least 95%
identical to SEQ ID NO:30 and a reverse primer at least 95% identical to
SEQ ID NO:31. In other examples, the pair of primers is specific for the
amplification of an influenza subtype European H7 and includes a forward
primer at least 95% identical to SEQ ID NO:33 and a reverse primer at
least 95% identical to SEQ ID NO:34. In other examples, the pair of
primers is specific for the amplification of an influenza subtype Asian
H9 and includes a forward primer at least 95% identical to 95% identical
to SEQ ID NO:36 and a reverse primer at least 95% identical to SEQ ID
NO:38.

[0137] Although exemplary probes and primers are provided in SEQ ID
NOS:3-38, one skilled in the art will appreciate that the primer and/or
probe sequence can be varied slightly by moving the probes a few
nucleotides upstream or downstream from the nucleotide positions that
they hybridize to on the influenza nucleic acid, provided that the probe
and or primer is still specific for the influenza sequence, such as
specific for the type or subtype of the influenza sequence, for example
specific for SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. For
example, one of skill in the art will appreciate that by analyzing the
consensus sequences shown in FIGS. 9-17 that variations of the probes and
primers disclosed as SEQ ID NOS:3-38 can by made by "sliding" the probes
and/or primers a few nucleotides 5' or 3' from their positions, and that
such variation will still be specific for the influenza viral type and/or
subtype.

[0138] Also provided by the present application are probes and primers
that include variations to the nucleotide sequences shown in any of SEQ
ID NOS:3-38, as long as such variations permit detection of the influenza
nucleic acid, such as an influenza type or subtype. For example, a probe
or primer can have at least 95% sequence identity such as at least 96%,
at least 97%, at least 98%, at least 99% to a nucleic acid consisting of
the sequence shown in any of SEQ ID NOS:3-38. In such examples, the
number of nucleotides does not change, but the nucleic acid sequence
shown in any of SEQ ID NOS:3-38 can vary at a few nucleotides, such as
changes at 1, 2, 3, or 4 nucleotides, for example by changing the
nucleotides as shown in the tables presented in FIGS. 9-17.

[0139] The present application also provides probes and primers that are
slightly longer or shorter than the nucleotide sequences shown in any of
SEQ ID NOS:3-38, as long as such deletions or additions permit detection
of the desired influenza nucleic acid, such as an influenza type or
subtype. For example, a probe can include a few nucleotide deletions or
additions at the 5'- or 3'-end of the probe shown in any of SEQ ID
NOS:3-38, such as addition or deletion of 1, 2, 3, or 4 nucleotides from
the 5'- or 3'-end, or combinations thereof (such as a deletion from one
end and an addition to the other end). In such examples, the number of
nucleotides changes. One of skill in the art will appreciate that the
consensus sequences shown in FIGS. 9-17 (SEQ ID NOS:42-50) provide
sufficient guidance as to what additions and/or subtractions can be made,
while still maintaining specificity for the influenza viral type and/or
subtype.

Detection and Identification of Influenza

[0140] A major application of the influenza virus specific primers and
probes disclosed herein is for the detection, typing and subtyping of
influenza viruses in a sample, such as a biological sample obtained from
a subject that has or is suspected of having an influenza infection.
Thus, the disclosed methods can be used to diagnose if a subject has an
influenza infection and/or discriminate between the viral type and/or
subtype the subject is infected with.

[0141] Methods for the detection of influenza nucleic acids are disclosed,
for example to determine if a subject is infected with an influenza
virus. Methods also are provided for determining the type and/or subtype
of the influenza viral nucleic acid, for example to determine the type
and/or subtype of influenza virus a subject is infected with.

[0143] Detecting an influenza nucleic acid in a sample involves contacting
the sample with at least one of the influenza specific probes disclosed
herein that is capable of hybridizing to an influenza virus nucleic acid
under conditions of very high stringency (such as a nucleic acid probe
capable of hybridizing under very high stringency conditions to an
influenza nucleic acid sequence set forth as SEQ ID NOS:42-50, for
example a nucleic acid sequence at least 95% identical to the nucleotide
sequence set forth as one of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ
ID NO:19, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ
ID NO:38), and detecting hybridization between the influenza virus
nucleic acid and the probe. Detection of hybridization between the probe
influenza nucleic acid indicates the presence of the influenza nucleic
acid in the sample.

[0144] By using influenza type specific probes, the disclosed methods can
be used to detect the presence of influenza types in the sample. For
example, by contacting the sample with an influenza type A specific
probe, such as a probe capable of hybridizing under very high stringency
conditions to an influenza nucleic acid sequence set forth as SEQ ID
NO:42, for example a nucleic acid sequence of at least 95% identical to
SEQ ID NO: 8, and detecting the hybridization of the influenza type A
specific probe to the influenza nucleic acid, the presence of influenza
type A is detected. Alternatively, contacting the sample with a probe
specific for an influenza type B nucleic acid, such as a probe capable of
hybridizing under very high stringency conditions to an influenza nucleic
acid sequence set forth as SEQ ID NO:43, for example a nucleic acid
sequence of at least 95% identical to SEQ ID NO:29, and detecting the
hybridization between the probe and the influenza nucleic acid indicates
influenza type B is present. Thus, these disclosed methods can be used
discriminate between the presence of influenza type A or type B in a
sample.

[0145] The influenza subtype specific probes disclosed herein can be used
to detect the presence of and discriminate between influenza subtypes in
a sample. For example, contacting a sample with a probe specific for
influenza subtype H1, such as a probe capable of hybridizing under very
high stringency conditions to an influenza nucleic acid sequence set
forth as SEQ ID NO:44, for example a nucleic acid at least 95% identical
to the nucleotide sequence set forth as SEQ ID NO:11, and detecting the
hybridization between the probe and the influenza nucleic acid indicates
that influenza subtype H1 is present. In another example, contacting a
sample with a probe specific for influenza subtype H3, such as a probe
capable of hybridizing under very high stringency conditions to an
influenza nucleic acid sequence set forth as SEQ ID NO:45, for example a
nucleic acid at least 95% identical to the nucleotide sequence set forth
as SEQ ID NO: 14, and detecting the hybridization between the probe and
the influenza nucleic acid indicates the presence of influenza subtype
H3. In another example, contacting a sample with a probe specific for
influenza subtype H5, such as a probe capable of hybridizing under very
high stringency conditions to an influenza nucleic acid sequence set
forth as SEQ ID NO:46, for example a nucleic acid at least 95% identical
to the nucleotide sequence set forth as SEQ ID NO:19, and detecting the
hybridization between the probe and the influenza nucleic acid indicates
the presence of influenza subtype H5. In another example, contacting a
sample with a probe specific for influenza subtype H5, such as a probe
capable of hybridizing under very high stringency conditions to an
influenza nucleic acid sequence set forth as SEQ ID NO:47, for example a
nucleic acid at least 95% identical to the nucleotide sequence set forth
as SEQ ID NO:24, and detecting the hybridization between the probe and
the influenza nucleic acid indicates the presence of influenza subtype
H5. In another example, contacting a sample with a probe specific for
influenza subtype North American H7, such as a probe capable of
hybridizing under very high stringency conditions to an influenza nucleic
acid sequence set forth as SEQ ID NO:48, for example a nucleic acid at
least 95% identical to the nucleotide sequence set forth as SEQ ID NO:32,
and detecting the hybridization between the probe and the influenza
nucleic acid indicates the presence of influenza subtype North American
H7. In yet another example, contacting a sample with a probe specific for
influenza subtype European H7, such as a probe capable of hybridizing
under very high stringency conditions to an influenza nucleic acid
sequence set forth as SEQ ID NO:49, for example a nucleic acid at least
95% identical to the nucleotide sequence set forth as SEQ ID NO:35, and
detecting the hybridization between the probe and the influenza nucleic
acid indicates the presence of influenza subtype European H7. In still
another example, contacting a sample with a probe specific for influenza
subtype Asian H9, such as a probe capable of hybridizing under very high
stringency conditions to an influenza nucleic acid sequence set forth as
SEQ ID NO:50, for example a nucleic acid at least 95% identical to the
nucleotide sequence set forth as SEQ ID NO:38, and detecting the
hybridization between the probe and the influenza nucleic acid indicates
the presence of influenza subtype Asian H9.

[0146] In some embodiments, detecting the presence of an influenza nucleic
acid sequence in a sample includes the extraction of influenza RNA. RNA
extraction relates to releasing RNA from a latent or inaccessible form in
a virion, cell or sample and allowing the RNA to become freely available.
In such a state, it is suitable for effective detection and/or
amplification of the influenza nucleic acid. Releasing RNA may include
steps that achieve the disruption of virions containing viral RNA, as
well as disruption of cells that may harbor such virions. Extraction of
RNA is generally carried out under conditions that effectively exclude or
inhibit any ribonuclease activity that may be present. Additionally,
extraction of RNA may include steps that achieve at least a partial
separation of the RNA dissolved in an aqueous medium from other cellular
or viral components, wherein such components may be either particulate or
dissolved.

[0147] One of ordinary skill in the art will know suitable methods for
extracting RNA from a sample; such methods will depend upon, for example,
the type of sample in which the influenza RNA is found. For example, the
RNA may be extracted using guanidinium isothiocyanate, such as the
single-step isolation by acid guanidinium
isothiocyanate-phenol-chloroform extraction of Chomczynski et al. (Anal.
Biochem. 162:156-59, 1987). The sample can be used directly or can be
processed, such as by adding solvents, preservatives, buffers, or other
compounds or substances. Viral RNA can be extracted using standard
methods. For instance, rapid RNA preparation can be performed using a
commercially available kit (such as the Roche MagNA Pure Compact Nucleic
Acid Isolation Kit I, QIAAMP® Viral RNA Mini Kit, QIAAMP®
MinElute Virus Spin Kit or RNEASY® Mini Kit (QIAGEN); NUCLISENS®
NASBA Diagnostics (bioMerieux); MASTERPURE® Complete DNA and RNA
Purification Kit (EPICENTRE). Alternatively, an influenza virion may be
disrupted by a suitable detergent in the presence of proteases and/or
inhibitors of ribonuclease activity. Additional exemplary methods for
extracting RNA are found, for example, in World Health Organization,
Manual for the virological investigation of polio, World Health
Organization, Geneva, 2001.

[0148] In some embodiments, the probe is detectably labeled, either with
an isotopic or non-isotopic label; in alternative embodiments, the
influenza nucleic acid is labeled. Non-isotopic labels can, for instance,
comprise a fluorescent or luminescent molecule, or an enzyme, co-factor,
enzyme substrate, or hapten. The probe is incubated with a
single-stranded or double-stranded preparation of RNA, DNA, or a mixture
of both, and hybridization determined. In some examples the hybridization
results in a detectable change in signal such as in increase or decrease
in signal, for example from the labeled probe. Thus, detecting
hybridization comprises detecting a change in signal from the labeled
probe during or after hybridization relative to signal from the label
before hybridization.

[0149] In some embodiments, influenza nucleic acids present in a sample
are amplified prior to using a hybridization probe for detection. For
instance, it can be advantageous to amplify a portion of the influenza
nucleic acid, then detect the presence of the amplified influenza nucleic
acid. For example, to increase the number of nucleic acids that can be
detected, thereby increasing the signal obtained. Influenza specific
nucleic acid primers can be used to amplify a region that is at least
about 50, at least about 60, at least about 70, at least about 80 at
least about 90, at least about 100, at least about 200, or more base
pairs in length to produce amplified influenza specific nucleic acids.
Any nucleic acid amplification method can be used to detect the presence
of influenza in a sample. In one specific, non-limiting example,
polymerase chain reaction (PCR) is used to amplify the influenza nucleic
acid sequences. In other specific, non-limiting examples, real-time PCR,
reverse transcriptase-polymerase chain reaction (RT-PCR), real-time
reverse transcriptase-polymerase chain reaction (rt RT-PCR), ligase chain
reaction, or transcription-mediated amplification (TMA) is used to
amplify the influenza nucleic acid. In a specific example, the influenza
virus nucleic acid is amplified by rt RT-PCR. Techniques for nucleic acid
amplification are well-known to those of skill in the art.

[0150] Typically, at least two primers are utilized in the amplification
reaction, however it is envisioned that one primer can be utilized, for
example to reverse transcribe a single stranded nucleic acid such as a
single-stranded influenza RNA. Amplification of the influenza nucleic
acid involves contacting the influenza nucleic acid with one or more
primers that are capable of hybridizing to and directing the
amplification of an influenza nucleic acid (such as a nucleic acid
capable of hybridizing under very high stringency conditions to an
influenza nucleic acid set forth as SEQ NO:42-50, for example a primer
that is least 95% identical to the nucleotide sequence set forth as one
of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24,
SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38). In some
embodiments, the sample is contacted with at least one primer that is
specific for an influenza type or subtype, such as those disclosed
herein.

[0151] In some embodiments, the sample is contacted with at least one pair
of primers that include a forward and reverse primer that both hybridize
to an influenza nucleic acid specific for an influenza viral type and or
subtype, such as influenza type A, type B, subtype H3, H5, H7(North
American or European), or Asian H9. Examples of suitable primer pairs for
the amplification of influenza type and/or subtype specific nucleic acids
are described above.

[0152] Any type of thermal cycler apparatus can be used for the
amplification of the influenza nucleic acids and/or the determination of
hybridization. Examples of suitable apparatuses include a PTC-100®
Peltier Thermal Cycler (MJ Research, Inc.; San Francisco, Calif.), a
ROBOCYCLER® 40 Temperature Cycler (Stratagene; La Jolla, Calif.), or
a GENEAMP® PCR System 9700 (Applied Biosystems; Foster City, Calif.).
For real-time PCR, any type of real-time thermocycler apparatus can be
used. For example, a BioRad iCycler iQ®, LIGHTCYCLER® (Roche;
Mannheim, Germany), a 7700 Sequence Detector (Perkin Elmer/Applied
Biosystems; Foster City, Calif.), ABI® systems such as the 7000, 7500,
7700, or 7900 systems (Applied Biosystems; Foster City, Calif.), or an
MX4000®, MX3000® or MX3005® (Stratagene; La Jolla, Calif.), and
Cepheid SMARTCYCLER® can by used to amplify nucleic acid sequences in
real-time.

[0153] The amplified influenza nucleic acid, for example an influenza type
or subtype specific nucleic acid, can be detected in real-time, for
example by real-time PCR such as real-time RT-PCR, in order to determine
the presence, the identity, and/or the amount of an influenza type or
subtype specific nucleic acid in a sample. In this manner, an amplified
nucleic acid sequence, such as an amplified influenza nucleic acid
sequence, can be detected using a probe specific for the product
amplified from the influenza sequence of interest, such as an influenza
sequence that is specific for influenza type A, type B, subtype H1, H3,
H5, North America H7, European H7, and Asian H9. Detecting the amplified
product includes the use of labeled probes that are sufficiently
complementary and hybridize to the amplified nucleic acid sequence. Thus,
the presence, amount, and/or identity of the amplified product can be
detected by hybridizing a labeled probe, such as a fluorescently labeled
probe, complementary to the amplified product. In one embodiment, the
detection of a target nucleic acid sequence of interest includes the
combined use of PCR amplification and a labeled probe such that the
product is measured using real-time RT-PCR. In another embodiment, the
detection of an amplified target nucleic acid sequence of interest
includes the transfer of the amplified target nucleic acid to a solid
support, such as a blot, for example a Northern blot, and probing the
blot with a probe, for example a labeled probe, that is complementary to
the amplified target nucleic acid sequence. In yet another embodiment,
the detection of an amplified target nucleic acid sequence of interest
includes the hybridization of a labeled amplified target nucleic acid to
probes disclosed herein that are an arrayed in a predetermined array with
an addressable location and that are complementary to the amplified
target nucleic acid.

[0154] In one embodiment, the fluorescently-labeled probes rely upon
fluorescence resonance energy transfer (FRET), or in a change in the
fluorescence emission wavelength of a sample, as a method to detect
hybridization of a DNA probe to the amplified target nucleic acid in
real-time. For example, FRET that occurs between fluorogenic labels on
different probes (for example, using HybProbes) or between a fluorophore
and a non-fluorescent quencher on the same probe (for example, using a
molecular beacon or a TAQMAN® probe) can identify a probe that
specifically hybridizes to the DNA sequence of interest and in this way,
using Influenza type and/or subtype specific probes, can detect the
presence, identity, and/or amount of an influenza type and/or subtype in
a sample. In one embodiment, the fluorescently-labeled DNA probes used to
identify amplification products have spectrally distinct emission
wavelengths, thus allowing them to be distinguished within the same
reaction tube.

[0155] In another embodiment, a melting curve analysis of the amplified
target nucleic acid can be performed subsequent to the amplification
process. The Tm of a nucleic acid sequence depends on the length of
the sequence and its G/C content. Thus, the identification of the Tm
for a nucleic acid sequence can be used to identify the amplified nucleic
acid.

Influenza Profiling Arrays

[0156] An array containing a plurality of heterogeneous probes for the
detection, typing, and/or subtyping of influenza viruses are disclosed.
Such arrays may be used to rapidly detect and/or identify the type and/or
subtype of an influenza virus in a sample. For example the arrays can be
used to determine the presence of influenza A or influenza B in a sample
and to determine if the influenza virus is of subtype H1, H3, H5,
H7(North American or European), or Asian H9.

[0157] Arrays are arrangements of addressable locations on a substrate,
with each address containing a nucleic acid, such as a probe. In some
embodiments, each address corresponds to a single type or class of
nucleic acid, such as a single probe, though a particular nucleic acid
may be redundantly contained at multiple addresses. A "microarray" is a
miniaturized array requiring microscopic examination for detection of
hybridization. Larger "macroarrays" allow each address to be recognizable
by the naked human eye and, in some embodiments, a hybridization signal
is detectable without additional magnification. The addresses may be
labeled, keyed to a separate guide, or otherwise identified by location.

[0158] In some embodiments, an influenza profiling array is a collection
of separate probes at the array addresses. The influenza profiling array
is then contacted with a sample suspected of containing influenza nucleic
acids under conditions allowing hybridization between the probe and
nucleic acids in the sample to occur. Any sample potentially containing,
or even suspected of containing, influenza nucleic acids may be used,
including nucleic acid extracts, such as amplified or non-amplified DNA
or RNA preparations. A hybridization signal from an individual address on
the array indicates that the probe hybridizes to a nucleotide within the
sample. This system permits the simultaneous analysis of a sample by
plural probes and yields information identifying the influenza nucleic
acids contained within the sample. In alternative embodiments, the array
contains influenza nucleic acids and the array is contacted with a sample
containing a probe. In any such embodiment, either the probe or the
influenza nucleic acids may be labeled to facilitate detection of
hybridization.

[0159] The nucleic acids may be added to an array substrate in dry or
liquid form. Other compounds or substances may be added to the array as
well, such as buffers, stabilizers, reagents for detecting hybridization
signal, emulsifying agents, or preservatives.

[0160] In certain examples, the array includes one or more molecules or
samples occurring on the array a plurality of times (twice or more) to
provide an added feature to the array, such as redundant activity or to
provide internal controls.

[0161] Within an array, each arrayed nucleic acid is addressable, such
that its location may be reliably and consistently determined within the
at least the two dimensions of the array surface. Thus, ordered arrays
allow assignment of the location of each nucleic acid at the time it is
placed within the array. Usually, an array map or key is provided to
correlate each address with the appropriate nucleic acid. Ordered arrays
are often arranged in a symmetrical grid pattern, but nucleic acids could
be arranged in other patterns (for example, in radially distributed
lines, a "spokes and wheel" pattern, or ordered clusters). Addressable
arrays can be computer readable; a computer can be programmed to
correlate a particular address on the array with information about the
sample at that position, such as hybridization or binding data, including
signal intensity. In some exemplary computer readable formats, the
individual samples or molecules in the array are arranged regularly (for
example, in a Cartesian grid pattern), which can be correlated to address
information by a computer.

[0162] An address within the array may be of any suitable shape and size.
In some embodiments, the nucleic acids are suspended in a liquid medium
and contained within square or rectangular wells on the array substrate.
However, the nucleic acids may be contained in regions that are
essentially triangular, oval, circular, or irregular. The overall shape
of the array itself also may vary, though in some embodiments it is
substantially flat and rectangular or square in shape.

[0163] Influenza profiling arrays may vary in structure, composition, and
intended functionality, and may be based on either a macroarray or a
microarray format, or a combination thereof. Such arrays can include, for
example, at least 10, at least 25, at least 50, at least 100, or more
addresses, usually with a single type of nucleic acid at each address. In
the case of macroarrays, sophisticated equipment is usually not required
to detect a hybridization signal on the array, though quantification may
be assisted by standard scanning and/or quantification techniques and
equipment. Thus, macroarray analysis as described herein can be carried
out in most hospitals, agricultural and medial research laboratories,
universities, or other institutions without the need for investment in
specialized and expensive reading equipment.

[0164] Examples of substrates for the arrays disclosed herein include
glass (e.g., functionalized glass), Si, Ge, GaAs, GaP, SiO2,
SiN4, modified silicon nitrocellulose, polyvinylidene fluoride,
polystyrene, polytetrafluoroethylene, polycarbonate, nylon, fiber, or
combinations thereof. Array substrates can be stiff and relatively
inflexible (for example glass or a supported membrane) or flexible (such
as a polymer membrane). One commercially available product line suitable
for probe arrays described herein is the Microlite line of
MICROTITER® plates available from Dynex Technologies UK (Middlesex,
United Kingdom), such as the Microlite 1+96-well plate, or the 384
Microlite+384-well plate.

[0165] Addresses on the array should be discrete, in that hybridization
signals from individual addresses can be distinguished from signals of
neighboring addresses, either by the naked eye (macroarrays) or by
scanning or reading by a piece of equipment or with the assistance of a
microscope (microarrays).

[0166] Addresses in an array may be of a relatively large size, such as
large enough to permit detection of a hybridization signal without the
assistance of a microscope or other equipment. Thus, addresses may be as
small as about 0.1 mm across, with a separation of about the same
distance. Alternatively, addresses may be about 0.5, 1, 2, 3, 5, 7, or 10
mm across, with a separation of a similar or different distance. Larger
addresses (larger than 10 mm across) are employed in certain embodiments.
The overall size of the array is generally correlated with size of the
addresses (for example, larger addresses will usually be found on larger
arrays, while smaller addresses may be found on smaller arrays). Such a
correlation is not necessary, however.

[0167] The arrays herein may be described by their densities (the number
of addresses in a certain specified surface area). For macroarrays, array
density may be about one address per square decimeter (or one address in
a 10 cm by 10 cm region of the array substrate) to about 50 addresses per
square centimeter (50 targets within a 1 cm by 1 cm region of the
substrate). For microarrays, array density will usually be one or more
addresses per square centimeter, for instance, about 50, about 100, about
200, about 300, about 400, about 500, about 1000, about 1500, about
2,500, or more addresses per square centimeter.

[0168] The use of the term "array" includes the arrays found in DNA
microchip technology. As one, non-limiting example, the probes could be
contained on a DNA microchip similar to the GENECHIP® products and
related products commercially available from Affymetrix, Inc. (Santa
Clara, Calif.). Briefly, a DNA microchip is a miniaturized, high-density
array of probes on a glass wafer substrate. Particular probes are
selected, and photolithographic masks are designed for use in a process
based on solid-phase chemical synthesis and photolithographic fabrication
techniques similar to those used in the semiconductor industry. The masks
are used to isolate chip exposure sites, and probes are chemically
synthesized at these sites, with each probe in an identified location
within the array. After fabrication, the array is ready for
hybridization. The probe or the nucleic acid within the sample may be
labeled, such as with a fluorescent label and, after hybridization, the
hybridization signals may be detected and analyzed.

Kits

[0169] The nucleic acid primers and probes disclosed herein can be
supplied in the form of a kit for use in the detection, typing, and/or
subtyping of influenza, including kits for any of the arrays described
above. In such a kit, an appropriate amount of one or more of the nucleic
acid probes and or primers is provided in one or more containers or held
on a substrate. A nucleic acid probe and/or primer may be provided
suspended in an aqueous solution or as a freeze-dried or lyophilized
powder, for instance. The container(s) in which the nucleic acid(s) are
supplied can be any conventional container that is capable of holding the
supplied form, for instance, microfuge tubes, ampoules, or bottles. The
kits can include either labeled or unlabeled nucleic acid probes for use
in detection, typing, and subtyping of influenza nucleotide sequences.

[0170] In some applications, one or more primers (as described above),
such as pairs of primers, may be provided in pre-measured single use
amounts in individual, typically disposable, tubes or equivalent
containers. With such an arrangement, the sample to be tested for the
presence of influenza nucleic acids can be added to the individual tubes
and amplification carried out directly.

[0171] The amount of nucleic acid primer supplied in the kit can be any
appropriate amount, and may depend on the target market to which the
product is directed. For instance, if the kit is adapted for research or
clinical use, the amount of each nucleic acid primer provided would
likely be an amount sufficient to prime several PCR amplification
reactions. General guidelines for determining appropriate amounts may be
found in Innis et al., Sambrook et al., and Ausubel et al. A kit may
include more than two primers in order to facilitate the PCR
amplification of a larger number of influenza nucleotide sequences.

[0172] In some embodiments, kits also may include the reagents necessary
to carry out PCR amplification reactions, including DNA sample
preparation reagents, appropriate buffers (such as polymerase buffer),
salts (for example, magnesium chloride), and deoxyribonucleotides
(dNTPs).

[0173] One or more control sequences for use in the PCR reactions also may
be supplied in the kit (for example, for the detection of human RNAse P).

[0174] Particular embodiments include a kit for detecting and typing
and/or subtyping an influenza nucleic acid based on the arrays described
above. Such a kit includes at least one probe specific for an influenza
nucleic acid (as described above) and instructions. A kit may contain
more than one different probe, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 20, 25, 50, 100, or more probes. The instructions may
include directions for obtaining a sample, processing the sample,
preparing the probes, and/or contacting each probe with an aliquot of the
sample. In certain embodiments, the kit includes an apparatus for
separating the different probes, such as individual containers (for
example, microtubules) or an array substrate (such as, a 96-well or
384-well microtiter plate). In particular embodiments, the kit includes
prepackaged probes, such as probes suspended in suitable medium in
individual containers (for example, individually sealed EPPENDORF®
tubes) or the wells of an array substrate (for example, a 96-well
microtiter plate sealed with a protective plastic film). In other
particular embodiments, the kit includes equipment, reagents, and
instructions for extracting and/or purifying nucleotides from a sample.

Synthesis of Oligonucleotide Primers and Probes

[0175] In vitro methods for the synthesis of oligonucleotides are well
known to those of ordinary skill in the art; such methods can be used to
produce primers and probes for the disclosed methods. The most common
method for in vitro oligonucleotide synthesis is the phosphoramidite
method, formulated by Letsinger and further developed by Caruthers
(Caruthers et al., Chemical synthesis of deoxyoligonucleotides, in
Methods Enzymol. 154:287-313, 1987). This is a non-aqueous, solid phase
reaction carried out in a stepwise manner, wherein a single nucleotide
(or modified nucleotide) is added to a growing oligonucleotide. The
individual nucleotides are added in the form of reactive
3'-phosphoramidite derivatives. See also, Gait (Ed.), Oligonucleotide
Synthesis. A practical approach, IRL Press, 1984.

[0176] In general, the synthesis reactions proceed as follows: A
dimethoxytrityl or equivalent protecting group at the 5' end of the
growing oligonucleotide chain is removed by acid treatment. (The growing
chain is anchored by its 3' end to a solid support such as a silicon
bead.) The newly liberated 5' end of the oligonucleotide chain is coupled
to the 3'-phosphoramidite derivative of the next deoxynucleotide to be
added to the chain, using the coupling agent tetrazole. The coupling
reaction usually proceeds at an efficiency of approximately 99%; any
remaining unreacted 5' ends are capped by acetylation so as to block
extension in subsequent couplings. Finally, the phosphite triester group
produced by the coupling step is oxidized to the phosphotriester,
yielding a chain that has been lengthened by one nucleotide residue. This
process is repeated, adding one residue per cycle. See, for example, U.S.
Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679, and 5,132,418.
Oligonucleotide synthesizers that employ this or similar methods are
available commercially (for example, the PolyPlex oligonucleotide
synthesizer from Gene Machines, San Carlos, Calif.). In addition, many
companies will perform such synthesis (for example, Sigma-Genosys, The
Woodlands, Tex.; Qiagen Operon, Alameda, Calif.; Integrated DNA
Technologies, Coralville, Iowa; and TriLink BioTechnologies, San Diego,
Calif.).

[0177] The following examples are provided to illustrate particular
features of certain embodiments. However, the particular features
described below should not be construed as limitations on the scope of
the invention, but rather as examples from which equivalents will be
recognized by those of ordinary skill in the art.

EXAMPLES

Example 1

Sample Collection and Preparation

[0178] This example describes exemplary procedures for the collection and
preparation of samples for the determination of the presence of influenza
nucleic acids.

[0179] Samples obtained from the respiratory tract were collected either
as broncheoalveolar lavage, tracheal aspirates, sputum, nasopharyngeal or
oropharyngeal aspirates or washes, or nasopharyngeal or oropharyngeal
swabs. Swabs were collected using swabs with a DACRON® tip and an
aluminum or plastic shaft. For specific viral isolates, viruses were
propagated in either MDCK cells or embryonated chicken eggs. For
validation of the primers and probes disclosed herein the following viral
isolates were used: X31 (H3N2) (Aichi/2/68×PR8 reassortant),
A/Panama/2007/99 (H3N2), A/New Calcdonia/20/99 (H1N1), ANietnam/1203/2003
(H5N1), A/HongKong/1203/99 (H9N2), A/Netherlands/219/2003 (H7N7), A/New
York/5295/2003 (H7N2) and B/Hong Kong/330/2001. Samples were refrigerated
or frozen prior to nucleic acid extraction. Viral RNA was extracted from
the samples using the QIAAMP® Viral RNAEASY® Mini Kit available
from QIAGEN® (Valencia, Calif.) according to the manufacturer's
recommendations.

Example 2

Selection of Probe/Primer Sets

[0180] This example describes the rational and procedures used to design
probes and primers for the detection, typing and subtyping of influenza
virus.

[0181] Oligonucleotide primers and probes for universal detection of
influenza type A and influenza type B influenza viruses were selected
from highly conserved (consensus) regions of the M and NS genes,
respectively, based on nucleotide alignments of all available sequence
data from GENBANK® database of National Centers for Biological
Information, NIH (NCBI) and the Influenza Sequence Database of Los Alamos
National Laboratories (LANL). Similarly, primers and probes specific for
the hemagglutinin (HA) gene of modern human H1, H3, Asian avian H5, North
American avian H7, European avian H7 and Asian avian H9 viruses were
designed. Because of the pandemic potential of influenza subtype Asian
avian H5, two redundant primer and probe set were designed to detect this
influenza subtype. The consensus sequence for the region of the influenza
type A M gene used for the design of probes and primers specific for
influenza type A is given in the table shown in FIGS. 9A-9F. The
consensus sequence for the region of the influenza type B NS gene used
for the design of probes and primers specific for influenza type B is
given in the table shown in FIGS. 10A-10I. The consensus sequences for
the region of the influenza subtype H1 HA gene used for the design of
probes and primers specific for influenza subtype H1 is given in the
table shown in FIGS. 11A-11F. The consensus sequences for the region of
the influenza subtype H3 HA gene used for the design of probes and
primers specific for influenza subtype H3 is given in the table shown in
FIGS. 12A-12F. The consensus sequence for a region of the influenza
subtype H5 HA gene used for the design of probes and primers specific for
influenza t subtype H5 is given in the table shown in FIGS. 13A-131. The
consensus sequence for a region of the influenza subtype H5 HA gene used
for the design of probes and primers specific for influenza subtype H5 is
given in the table shown in FIGS. 14A-14L. The consensus sequence for the
region of the influenza subtype North American H7 HA gene used for the
design of probes and primers specific for influenza subtype North
American H7 is given in the table shown in FIGS. 15A-15B. The consensus
sequence for the region of the influenza subtype European H7 HA gene used
for the design of probes and primers specific for influenza subtype
European H7 is given in the table shown in FIGS. 16A-16C. The consensus
sequence for the region of the influenza subtype Asian H9 HA gene used
for the design of probes and primers specific for influenza subtype Asian
H9 is given in the table shown in FIGS. 17A-17I. With reference to FIGS.
9-17 the consensus sequence for the influenza viral type or subtype
specific nucleic acid is shown at the top of each table. The boxed
sequences represent the positions of exemplary probes and primers
disclosed herein. Nucleotide variations for the indicated influenza viral
isolates are shown in the columns below the consensus sequence, with a
dot meaning that the nucleotide present at that position is identical to
the consensus sequence. In addition K=G or T; S=G or C; R=A or G; M=A or
C; and Y=T or C.

[0182] In order to avoid loss of reaction performance due to primer-dimer
or hairpin loop formation, primers and probes were evaluated using
Software packages PRIMEREXPRESS® (Applied Biosystems) and BEACON
DESIGNER 4.0® (PREMIER Biosoft International) to predict secondary
structures and self-annealing probabilities.

[0183] Each primer and probe sequence was subjected to a nucleotide Blast
search (NCBI) against the entire GENBANK® nucleotide database to
validate their specificities and avoid non-specific reactivity. The probe
and primers listed in Table 1 were selected for validation using
TAQMAN® chemistry. Primers and dual-labeled TAQMAN® probes (Table
1) were synthesized by the Biotechnology Core Facility, Centers for
Disease Control.

[0184] The reaction efficiency of the primer sets was individually tested
in a set of five-fold serial dilutions of viral RNA using SYBER green
binding to double stranded nucleic acids as an indicator of
amplification. All it RT-PCR assays for detection and characterization of
influenza were designed to achieve reaction efficiencies of approximately
100%. A reaction efficiency of 100% indicates that a primer set is
capable of achieving a complete doubling of the nucleic acid target
sequence in a single round of amplification.

[0185] With reference to FIG. 5A-5C, the reaction efficiency of the primer
set for universal detection of influenza type A was determined by testing
against a five-fold serial dilution of viral RNA. Identical tests were
carried out with the primer sets specific for each viral type and
subtype. FIG. 5 A shows the relative fluorescence of SYBER green when
bound to double stranded nucleic acid versus the number of PCR cycles.
The individual it RT-PCR reactions were subjected to melting curve
analysis to confirm that the SYBER green fluorescence was attributable to
specific amplification of the influenza A gene target. As shown in FIG.
5B, all reactions showed double stranded nucleic acid melting at the same
temperature, indicating specific amplification. Similar melting curve
analysis was performed for all primer sets and demonstrated that the
primers were specific for their specific target influenza nucleic acid
sequence. As shown on FIG. 5C, reaction Ct values for the influenza A
specific primers were plotted against their relative RNA concentration
and the doubling efficiency (% reaction efficiency) was determined by
estimating the slope using regression analysis. A slope of 3.23 indicates
a reaction efficiency of approximately 100%. A reaction efficiency of
100.3% percent was obtained for the influenza type A specific primers.
All primer sets tested had a reaction efficiency of approximately 100%
when subjected to the same analysis.

[0186] Following the validation of the reaction specificity and efficiency
of the primer sets the reaction efficiency of the primer/probe sets was
validated. Using a five-fold viral dilutions series the reaction
efficiency of the individual influenza type and subtype primer/probe sets
was analyzed. Exemplary data for the analysis of the probe/primer set
specific for influenza type A is shown in FIG. 6A and FIG. 6B.

[0187] As shown in FIG. 6A, the reaction efficiency of the primer/probe
set for universal detection of type A influenza was determined by testing
against a five-fold serial dilution of viral RNA. The reaction Ct values
were plotted against their relative RNA concentration to estimate the
reaction efficiency using regression analysis (FIG. 6B). Similar test
were carried out on all available primer sets. As shown in Table 2, all
of the primer/probe sets exhibited reaction efficiencies at or near 100%.

[0188] One of the design criteria for the disclosed primer and probe sets
was that they could be used at a variety of annealing (hybridization)
temperatures. Thus, the probe/primer sets were tested for their ability
to perform at a range of annealing temperatures from 50-62.5° C.
FIG. 7 shows a plot of the real-time RT-PCR reactivity comparison of the
influenza A primer/probe set with annealing temperatures ranging from
50-62.5° C. In order to determine the optimal thermocycling
conditions, each probe/primer set was similarly tested with annealing
temperatures ranging from 50-62.5° C. All primer/probe sets were
designed to demonstrate comparable reactivity at annealing temperatures
ranging from 50-60° C. and exhibited stable Ct values at all
temperatures tested (Table 3 and Table 4).

[0191] Individual 1.5 ml microcentrifuge tubes were prepared for each
individual primer/probe set used. Individual primers and probes were
vortexed and briefly centrifuged prior to dispensing. Into each
microcentrifuge tube was added 20 microliter rt RT-PCR master mix,
wherein the master mix was optimized for various real time PCR
instruments. The mister mix was prepared as shown in Table 5.

Where N is the number of samples including non template controls (NTC).
For viral template controls (VTC) and positive controls for human RNAse P
individual mastermixes were prepared. The reactions were mixed by
pipeting up and down, without vortexing. Twenty microliters of each
master mix was added into individual wells of a 96 well plate. An example
of the arrayed format used is shown in Table 6 below:

FluA is a primer probe set specific for influenza A; H1 is a primer probe
set specific for H1; H3 is a primer probe set specific for H3; H5a is a
primer probe set specific for H5; H5b is a primer probe set specific for
H5; H9 is a primer probe set specific for H9; FluB is a primer probe set
specific for influenza B; and RNP is a primer probe set specific for
human RNAse P.

[0192] RNA Samples of viral unknown as well as the NTC, VTC, and a mock
extraction control were added to the individual wells. NTCs were added
first to control for contamination in the master mix. For NTCs 5
microliters of distilled water was added. Five microliters of viral
unknown was added to each well with the exception of control wells. For
positive controls five microliters of viral RNA was added. The mock
extraction controls were added after the samples have been added to
control for cross-contamination during sample preparation or addition.
VTCs were added last after all samples and NTCs were sealed to prevent
contamination. An example of the array format used is shown in Table 7
below:

Where NTC is the non-template control (no RNA). S1-S9 are samples
obtained from a subject(s). MOCK is a mock extraction control and VTC is
the viral template control.

Example 4

Real Time RT-PCR of Samples

[0193] This example describes rt-RT-PCR parameters used for the
determination of the presence, type and subtype of influenza in a sample.

[0194] Prior to an rt RT-PCR run, the 96 well plate was centrifuged at
500×g for 30 seconds at 4° C. The plate was loaded into a
thermocycler and subjected to the PCR cycle as shown in Table 8. All
reactions were performed on a Stratagene MX4000®, MX3000P® or
BioRad IQ ICYCLER® platform. PCR conditions were optimized for each of
the listed instruments. The reaction volume was 25 μl.

The Determination of Influenza Viral Type and Subtype in Samples Obtained
from Subjects

[0195] This example describes the determination of the presence, type, and
subtype of influenza viral nucleic acid in samples obtained from
subjects.

[0196] Samples obtained from four subjects were tested for the presence of
influenza using influenza specific probe and primer sets disclosed herein
in rt RT-PCR TAQMAN® assays. In addition, the samples were tested for
the presence of influenza viral types A and B and influenza subtypes H1,
H2, and H5. The detection of human RNAse P was used as a control.

[0197] The rt RT-PCR data obtained for samples 1, 2, and 3 is shown in
FIGS. 8A, 8B, and 8C respectively. FIG. 8A shows the rt RT-PCR runs for
sample 1, which was determined to be positive for influenza type A
subtype H5. FIG. 8B shows the rt RT-PCR runs for sample 2, which was
determined to be positive for influenza type A subtype H3. FIG. 8C shows
the rt RT-PCR runs for sample 3s, which was determined to not contain
influenza.

[0198] While this disclosure has been described with an emphasis upon
particular embodiments, it will be obvious to those of ordinary skill in
the art that variations of the particular embodiments may be used, and it
is intended that the disclosure may be practiced otherwise than as
specifically described herein. Features, characteristics, compounds,
chemical moieties, or examples described in conjunction with a particular
aspect, embodiment, or example of the invention are to be understood to
be applicable to any other aspect, embodiment, or example of the
invention. Accordingly, this disclosure includes all modifications
encompassed within the spirit and scope of the disclosure as defined by
the following claims.